Mechanisms of failed apoptotic cell clearance by phagocyte subsets in cardiovascular disease

Recent evidence in humans indicate that defective phagocytic clearance of dying cells is linked to progression of advanced atherosclerotic lesions, the precursor to atherothrombosis, ischemic heart disease, and leading cause of death in the industrialized world. During atherogenesis, apoptotic cell turnover in the vascular wall is counterbalanced by neighboring phagocytes with high clearance efficiency, thereby limiting cellularity and maintaining lesion integrity. However, as lesions mature, phagocytic removal of apoptotic cells (efferocytosis) becomes defective, leading to secondary necrosis, expansion of plaque necrotic cores, and susceptibility to rupture. Recent genetic causation studies in experimental rodents have implicated key molecular regulators of efferocytosis in atherosclerotic progression. These include MER tyrosine kinase (MERTK), milk fat globule-EGF factor 8 (MFGE8), and complement C1q. At the cellular level, atheromata are infiltrated by a heterogenous population of professional phagocytes, comprised of monocytes, differentiated macrophages, and CD11c⁺ dendritic-like cells. Each cell type is characterized by disparate clearance efficiencies and varying activities of key phagocytic signaling molecules. It is in this context that we outline a working model whereby plaque necrosis and destabilization is jointly promoted by (1) direct inhibition of core phagocytic signaling pathways and (2) expansion of phagocyte subsets with poor clearance capacity. Towards identifying targets for promoting efficient apoptotic cell clearance and resolving inflammation in atherosclerosis and during ischemic heart disease and post myocardial infarction, this review will discuss potential in vivo suppressors of efferocytosis at each stage of clearance and how these putative interventional targets may differentially affect uptake at the level of vascular phagocyte subsets.     

Calcium-mediated pore expansion and cell death following nanoelectroporation

Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca^2 + after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca^2 + level from the nominal 2–5 μM to 2 mM for the first 60–90 min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca^2 +, the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2 mM Ca^2 + in the medium. With the nominal Ca^2 +, most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2 mM Ca^2 +, the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca^2 +-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture.     

Injury due to extravasation of thiopental and propofol: Risks/effects of local cooling/warming in rats

Inadvertent leakage of medications with vesicant properties can cause severe necrosis in tissue, which can have devastating long-term consequences. The aim of this study was to evaluate the extent of extravasation injury induced by thiopental and propofol, and the effects of cooling or warming of local tissue on extravasation injury at macroscopic and histopathologic levels. Rats were administered intradermally thiopental (2.5 mg/100 µL) or propofol (1.0 mg/100 µL). Rats were assigned randomly to three groups: control (no treatment), cooling and warming. Local cooling (18–20 °C) or warming (40–42 °C) was applied for 3 h immediately after agent injection. Lesion sizes (erythema, induration, ulceration, necrosis) were monitored after agent injection. Histopathology was evaluated in skin biopsies taken 24 h after agent injection. Thiopental injection induced severe skin injury with necrosis. Peak lesions developed within 24 h and healed gradually 18–27 days after extravasation. Propofol induced inflammation but no ulceration, and lesions healed within 1–2 days. Local cooling reduced thiopental- and propofol-induced extravasation injuries but warming strongly exacerbated the skin lesions (e.g., degeneration, necrosis) induced by extravasation of thiopental and propofol. Thiopental can be classified as a “vesicant” that causes tissue necrosis and propofol can be classified as an “irritant”. Local cooling protects (at least in part) against skin disorders induced by thiopental and propofol, whereas warming is harmful.     

The mechanism underlying the appearance of late apoptotic neutrophils and subsequent TNF-α production at a late stage during Staphylococcus aureus bioparticle-induced peritoneal inflammation in inducible NO synthase-deficient mice

During inflammation, neutrophils infiltrate into the involved site and undergo apoptosis. Early apoptotic neutrophils are then cleared by phagocytes, leading to resolution of the inflammation, whereas if late apoptotic neutrophils are accumulated for some reason, they provoke proinflammatory responses such as TNF-α production. To determine how endogenously produced nitric oxide (NO) regulates neutrophil apoptosis and the resolution of inflammation, we compared peritoneal inflammation induced by Staphylococcus aureus bioparticles in wild type mice with that in inducible NO synthase (iNOS)-deficient ones. In this model, NO production was largely dependent on iNOS, the NO level peaking at 24 h. There were increases in the numbers of neutrophils and late apoptotic ones at 24 h in iNOS-deficient mice as compared with in wild type ones, and consequently TNF-α production at 36 h in iNOS-deficient mice. On the other hand, the administration of a NO donor to iNOS-deficient mice at 12 h decreased the numbers of neutrophils and late apoptotic ones at 24 h, and thereafter TNF-α production at 36 h. In addition, coculturing of macrophages with late apoptotic neutrophils caused TNF-α production and a NO donor inhibited the transmigration of neutrophils in a dose-dependent manner. Collectively, these results suggest a novel mechanism that endogenously produced NO suppresses neutrophil accumulation at a late stage of inflammation, thereby preventing the appearance of late apoptotic neutrophils and subsequent proinflammatory responses.     

Ligand-dependent kinase activity of MERTK drives efferocytosis in human iPSC-derived macrophages

Removal of apoptotic cells by phagocytes (also called efferocytosis) is a crucial process for tissue homeostasis. Professional phagocytes express a plethora of surface receptors enabling them to sense and engulf apoptotic cells, thus avoiding persistence of dead cells and cellular debris and their consequent effects. Dysregulation of efferocytosis is thought to lead to secondary necrosis and associated inflammation and immune activation. Efferocytosis in primarily murine macrophages and dendritic cells has been shown to require TAM RTKs, with MERTK and AXL being critical for clearance of apoptotic cells. The functional role of human orthologs, especially the exact contribution of each individual receptor is less well studied. Here we show that human macrophages differentiated in vitro from iPSC-derived precursor cells express both AXL and MERTK and engulf apoptotic cells. TAM RTK agonism by the natural ligand growth-arrest specific 6 (GAS6) significantly enhanced such efferocytosis. Using a newly-developed mouse model of kinase-dead MERTK, we demonstrate that MERTK kinase activity is essential for efferocytosis in peritoneal macrophages in vivo. Moreover, human iPSC-derived macrophages treated in vitro with blocking antibodies or small molecule inhibitors recapitulated this observation. Hence, our results highlight a conserved MERTK function between mice and humans, and the critical role of its kinase activity in homeostatic efferocytosis.Subject terms: Immune cell death, Peritoneal macrophages     

RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes

Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1 μM fMLF and 1 μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells.     

A straightforward procedure to biosynthesise melatonin using freshly chopped Achillea millefolium L. as reagent

d, l-Tryptophan as the precursor of melatonin and Achillea millefolium L. as the plant cell tissue were used in order to force the plant enzymatic system to enhance the production of melatonin.The biotransformation protocol was performed by adding the precursor to the freshly chopped plant material suspended in water and stirred at room temperature in total darkness. The melatonin content was evaluated by LC-DAD-ESI-MS. The precursor-treated sample gave at least six times (345 ng/g_{fresh plant}) the melatonin amount usually produced in young plants of A. millefolium L. (50 ng/g_fresh plant).The present study shows that, in principle, adding a specific precursor to a suitable freshly chopped plant material can significantly enhance a secondary metabolite production.     

Casearin X exhibits cytotoxic effects in leukemia cells triggered by apoptosis

Clerodane diterpenes have demonstrated cytotoxic, antiplasmodial and anti-ulcer properties. In the present work, we determined the cytotoxic effect of casearin L (Cas L), O (Cas O) and X (Cas X) and (?)-hardwickiic acid isolated from Casearia sylvestris leaves, and investigated the underlying mechanisms involved in in vitro cell death induced by Cas X in HL-60 leukemia cells (0.7, 1.5 and 3.0 μM). Cytotoxicity tests demonstrated that Cas X was the most active compound studied, showing greater cytotoxic effects against CEM and HL-60 lines (IC₅₀ of 0.4 μM) and human peripheral blood mononuclear cells (PBMC, IC₅₀ of 1.2 μM). After 24 h exposure, Cas X caused a decrease in 5-bromo-20-deoxyuridine (BrdU) incorporation (36.6 and 24.5% labeling at 0.7 and 1.5 μM, respectively), reduction in viability, and increase in apoptotic and necrotic leukemia cells in a dose-dependent manner evidenced by the trypan blue and AO/EB (acridine orange/ethidium bromide) assays. Moreover, Cas X-treated cells exhibited nuclear fragmentation and cytoplasmic vacuolization depending on the concentration tested. These characteristics of apoptosis or secondary necrosis were confirmed by flow cytometry which revealed DNA fragmentation, phosphatidylserine externalization, activation of the effector caspases 3/7 and mitochondrial depolarization. We then found evidence that Cas X causes cell death via apoptotic pathways, corroborating the potential of casearins as compounds with promising antitumor-related properties.     

Antioxidant effects of captopril against lead acetate-induced hepatic and splenic tissue toxicity in Swiss albino mice

Considering that lead caused a lot of health problems in the world, the present study was carried out to investigate the protective effect of captopril as antioxidants to reduce liver and spleen toxicity induced by lead. Animals were divided into 3 groups, the 1st group served as control group, the 2nd group received 20 mg/kg of lead acetate and the 3rd group received 50 mg/kg of captopril one hour prior to lead administration for 5 days. Results showed that lead intake caused severe alterations in the liver and spleen manifested by hepatocytes degeneration, leukocytic infiltration, fibrosis in liver and moderate to severe liver pathological score. Spleen showed ill-defined architecture, presence of large macrophages and lymphoid necrosis. Administration of captopril reduced hepatotoxicity, liver fibrosis and decrease in pathological scoring system. Moreover, reduced toxicity in spleen is represented by reduction in necrotic areas, more or less healthy lymphoid follicles and decreasing in pathological scoring system.     

Oestrus in the Julia Creek dunnart (Sminthopsis douglasi) is associated with wheel running behaviour but not necessarily changes in body weight,food consumption or pouch morphology

The Julia Creek dunnart (Sminthopsis douglasi) is an endangered carnivorous marsupial belonging to the family Dasyuridae. This study investigated the oestrous cycle of this species in terms of its reproductive physiology and behaviour to explore more efficient methods of oestrus detection. Ten sexually mature captive female dunnarts were monitored daily at David Fleay Wildlife Park, Burleigh Heads, Australia, from mid September to late December 2006 for changes in urogenital cytology within the urine (0, 1+, 2+ and 3+), running wheel activity, body weight, uneaten food, faecal steroid metabolites (progesterone and oestradiol) and pouch development. Periods of increased running wheel activity were associated (p = 0.004) with an increase in the proportion of cornified urogenital epithelial cells found in the urine; periods of decreasing weight (p < 0.001) and uneaten food (p < 0.001) were also associated with changes in urogenital cytology but not to the point where they would be useful for oestrus detection. Between 60.3% and 92.0% of peak distances (confidence interval 95%) occurred when the epithelial cell index was 2+ or 3+. Only 15.5–37.5% of peak weights (CI: 95%) and 28.1–49.9% of incidences of uneaten food (CI: 95%) occurred when the epithelial cell index was 2+ or 3+. There was no significant difference in the mean length of the oestrous cycle when measured by urogenital cytology (mean ± SD: 25.0 ± 5.7 days; n = 20) or peak distance travelled (mean ± SD: 25.4 ± 5.7 days; n = 20). Changes in the concentration of oestradiol metabolites in Julia Creek Dunnart faeces were not useful in characterising the oestrous cycle. Wheel running activity declined markedly with increased faecal progestagen concentration. The majority of the pouch variables examined showed maximum development during the inter-oestrus period but as there was considerable variation between animals, the pouch was not considered a useful index of oestrus.     

Genetic polymorphisms involved in the inflammatory response and lung cancer risk: A case-control study in Japan

Evidence is accumulating that chronic inflammation may have an important mechanism for the development and progression of lung cancer. Therefore, genetic polymorphisms in genes that involved in the inflammatory response may be associated with lung cancer risk. We evaluated the role of tumor necrosis factor α (TNFA) rs1799724, interleukin 1β (IL1B) rs16944, IL6 rs1800796, myeloperoxidase (MPO) rs2333227 and C-reactive protein (CRP) rs2794520 in a case-control study comprised of 462 lung cancer cases and 379 controls in a Japanese population. Unconditional logistic regression was used to assess the adjusted odds ratios (OR) and 95% confidence intervals (95% CI). CRP rs2794520 (OR = 1.64, 95% CI = 1.19–2.26) and IL6 rs1800796 (OR = 1.41, 95% CI = 1.02–1.96) were associated with lung cancer risk. In addition, we assessed interactions between the polymorphisms and smoking. The polymorphisms did not significantly modify the association between smoking and lung cancer. As TNFA triggers a cytokine cascade, the modifying effect of the TNFA rs1799724 genotypes on the association of any of the remaining polymorphisms with lung cancer risk was also examined. There was a significant interaction between TNFA rs1799724 and MPO rs2333227 (P_interaction = 0.058). Future studies involving larger control and case populations will undoubtedly lead to a more thorough understanding of the role of the polymorphisms involved in the inflammation pathway in lung cancer.     

The effect of interleukins 27 and 35 and their role on mediating the action of insulin Like Growth Factor -1 on the inflammation and blood flow of chronically inflamed rat knee joint

IntroductionPrevious studies have shown that some cytokines mediate the effect of IGF-1 on inflammation and also association between IGF-1 and vascular endothelial dysfunction. Due to the discrepancies in the inflammatory and anti-inflammatory roles of IL-27 and IL-35, the effects of these cytokines and their IGF-1-mediating role were investigated regarding chronic joint inflammation and synovial blood flow.MethodMale rats were divided into two main groups of histopathology (n = 80) and blood flow (n = 72). These were further divided into ten subgroups of control, vehicle, IGF-1, IL-27, IL-35, their antagonists, IGF-1 + IL-27 antagonist, and IGF-1 + IL-35 antagonist. Inflammation was induced by intra-articular injection of complete Freund adjuvant. Two weeks later (in order to induce chronic inflammation), vehicle or drugs were injected into the joint space every other day until day 28, on which inflammatory indices were assessed histopathologically. In the second subgroups, vehicle or drugs were administered by super-fusion on day 28 and their effects on the joint blood flow (JBF, laser Doppler perfusion method) and the systemic blood pressure were assessed.ResultsEndogenous IL-27 and IL-35 had inflammatory roles and IGF-1 had no effect. IL-27 and IL-35 antagonists had the highest anti-inflammatory and anti-angiogenesis effects and these effects were inhibited by IGF-1. Total inflammation score was 4.5 ± 0.42, 3.50 ± 0.5, 2.25 ± 0.45 and 1.50 ± 0.42 for vehicle, IGF-1 antagonist, IL-27 antagonist and IL-35 antagonist respectively. A significant increase was induced in JBF by IGF-1 antagonist and combination of IGF-1 + IL-35 antagonist.ConclusionIL-27 and IL-35 antagonists may be suitable goals for the treatment of chronic joint inflammation while their anti-inflammatory effects are not exerted via the changes in JBF.     

Regulation and actions of activin A and follistatin in myocardial ischaemia–reperfusion injury

Activin A, a member of the transforming growth factor-β superfamily, is stimulated early in inflammation via the Toll-like receptor (TLR) 4 signalling pathway, which is also activated in myocardial ischaemia–reperfusion. Neutralising activin A by treatment with the activin-binding protein, follistatin, reduces inflammation and mortality in several disease models. This study assesses the regulation of activin A and follistatin in a murine myocardial ischaemia–reperfusion model and determines whether exogenous follistatin treatment is protective against injury. Myocardial activin A and follistatin protein levels were elevated following 30 min of ischaemia and 2 h of reperfusion in wild-type mice. Activin A, but not follistatin, gene expression was also up-regulated. Serum activin A did not change significantly, but serum follistatin decreased. These responses to ischaemia–reperfusion were absent in TLR4^−/− mice. Pre-treatment with follistatin significantly reduced ischaemia–reperfusion induced myocardial infarction. In mouse neonatal cardiomyocyte cultures, activin A exacerbated, while follistatin reduced, cellular injury after 3 h of hypoxia and 2 h of re-oxygenation. Neither activin A nor follistatin affected hypoxia-reoxygenation induced reactive oxygen species production by these cells. However, activin A reduced cardiomyocyte mitochondrial membrane potential, and follistatin treatment ameliorated the effect of hypoxia-reoxygenation on cardiomyocyte mitochondrial membrane potential. Taken together, these data indicate that myocardial ischaemia–reperfusion, through activation of TLR4 signalling, stimulates local production of activin A, which damages cardiomyocytes independently of increased reactive oxygen species. Blocking activin action by exogenous follistatin reduces this damage.     

Tumor Necrosis Factor gene polymorphism results in high TNF level in sepsis and septic shock

IntroductionSystemic sepsis releases several cytokines among which tumor necrosis factor alfa (TNFα) has emerged as key cytokine causing septic shock. Single Nucleotide Polymorphisms (SNPs) at positions ?238, ?308, ?376 and +489 in the promoter region of TNF gene exhibit differential association to inflammation and increased TNF production in sepsis.Materials and MethodsThis research work was carried out in 278 critically ill patients and 115 controls. The patients were divided into four groups: Healthy controls, SIRS, Sepsis and Septic shock. Plasma cytokine level was evaluated by ELISA. Specific sequences of TNF gene (?238, ?308, ?376, +489) were amplified using polychromase chain reaction (PCR). SNP detected by BamHiI, NcoI, FokI, TaiI restriction enzymes.ResultsMean plasma TNFα level in healthy Control group was 8.37 ± 2.23 pg/ml, in SIRS group, the mean plasma TNFα level was 77.99 ± 5.51 pg/ml, in Sepsis patients 187.1 ± 14.33 pg/ml and in septic shock 202.2 ± 14.85 pg/ml; range 56.17–417.1 pg/ml. SNP was studied among different patient groups, which showed a higher frequency of mutants among sepsis and shock patients as compared to control.ConclusionPlasma TNF alpha level was significantly high in patients with sepsis and septic shock. SNP of TNF gene showed significant association between polymorphism and development of severe sepsis and septic shock, this would help us in evaluating patients at high risk for septic shock and such patients needed to obtain a rational basis for therapy.     

Chronic vitamin E deficiency promotes vitamin C deficiency in zebrafish leading to degenerative myopathy and impaired swimming behavior

We hypothesized that zebrafish (Danio rerio) undergoing long-term vitamin E deficiency with marginal vitamin C status would develop myopathy resulting in impaired swimming. Zebrafish were fed for 1 y a defined diet without (E ?) and with (E +) vitamin E (500 mg α-tocopherol/kg diet). For the last 150 days, dietary ascorbic acid concentrations were decreased from 3500 to 50 mg/kg diet and the fish sampled periodically to assess ascorbic acid concentrations. The ascorbic acid depletion curves were faster in the E ? compared with E + fish (P < 0.0001); the estimated half-life of depletion in the E ? fish was 34 days, while in it was 55 days in the E + fish. To assess swimming behavior, zebrafish were monitored individually following a “startle-response” stimulus, using computer and video technology. Muscle histopathology was assessed using hematoxylin and eosin staining on paramedian sections of fixed zebrafish. At study end, E ? fish contained 300-fold less α-tocopherol (p < 0.0001), half the ascorbic acid (p = 0.0001) and 3-fold more malondialdehyde (p = 0.0005) than did E + fish. During the first minute following a tap stimulus (p < 0.05), E + fish swam twice as far as did E ? fish. In the E ? fish, the sluggish behavior was associated with a multifocal, polyphasic, degenerative myopathy of the skeletal muscle. The myopathy severity ranged from scattered acute necrosis to widespread fibrosis and was accompanied by increased anti-hydroxynonenal staining. Thus, vitamin E deficiency in zebrafish causes increased oxidative stress and a secondary depletion of ascorbic acid, resulting in severe damage to muscle tissue and impaired muscle function.     

Male infertility: Decreased levels of selenium,zinc and antioxidants

In this study, we aimed to compare the level of zinc, selenium, glutathione peroxidase activity and antioxidant status in following populations of men: severe inflammation in prostate (>10⁶ white blood cells in prostate secretion; n = 29), severe leukocytospermia, (>10⁶ white blood cells in semen; n = 31), mild inflammation, (0.2–1 M white blood cells in semen or prostate secretion; n = 24), non-inflammatory oligozoospermia (n = 32) and healthy controls (n = 27). Male partners of infertile couples had reduced level of antioxidative activity, selenium and zinc in their seminal plasma. Most importantly, reduced selenium levels were evident in all patient groups regardless of inflammation status. Therefore, these patients might gain some benefit from selenium supplementation.     

Effects of ezetimibe on markers of synthesis and absorption of cholesterol in high-risk patients with elevated C-reactive protein

AimsHigh-risk subjects with elevated C-reactive protein (CRP) are at high risk for cardiovascular events and frequently require potent statins or combined lipid-lowering therapy to achieve lipid targets and decrease inflammation. Our study aimed at evaluating the effects of three lipid-modifying therapies on LDL-cholesterol, CRP levels and markers of cholesterol absorption and synthesis.Main methodsA prospective intervention study was performed in high cardiovascular risk individuals receiving atorvastatin 10 mg daily for four weeks. Those with CRP ≥ 2.0 mg/L were randomized to another four-week treatment period with atorvastatin 40 mg, ezetimibe 10 mg or the combination of atorvastatin 40 mg / ezetimibe 10 mg. Lipids, markers of cholesterol absorption (campesterol and β-sitosterol), and synthesis (desmosterol), as well as CRP were quantified at baseline and end of study.Key findingsOne hundred and twenty two individuals were included. Atorvastatin alone or combined with ezetimibe reduced both LDL-cholesterol and CRP (P < 0.002 vs. baseline; Wilcoxon); ezetimibe did not modify CRP. Ezetimibe-based therapies reduced absorption markers and their ratios to cholesterol (P < 0.0001 vs. baseline, for all; Wilcoxon), whereas atorvastatin alone increased campesterol/cholesterol and β-sitosterol/cholesterol ratios (P < 0.05 vs. baseline; Wilcoxon). In addition, ezetimibe also increased desmosterol and desmosterol/cholesterol ratio (P < 0.0001 vs. baseline; Wilcoxon).SignificanceThese results contribute to understanding the link between cellular cholesterol homeostasis, inflammation and lipid-modifying therapies. Our findings highlight the broader benefit of combined therapy with a potent statin and ezetimibe decreasing inflammation, and preventing increase in cholesterol biosynthesis, an effect not observed with ezetimibe alone.     

TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca^2 + signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca^2 + entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP–BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20 ng/ml, 48 h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10 μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca^2 + (EGTA; 1 mM) or intracellular Ca^2 + (BAPTA; 5 μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca^2 + influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca^2 + and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.     

Inactivation of IL-6 and soluble IL-6 receptor by neutrophil derived serine proteases in cystic fibrosis

The ability of IL-6 to signal via both membrane bound and soluble receptors is thought to explain the capacity of this cytokine to act in both the initiation and resolution of acute inflammatory responses. In cystic fibrosis (CF), poorly resolved neutrophillic inflammation of the lungs is a primary cause of morbidity and mortality. Expression of IL-6 has been reported to be low in CF lung secretions, despite ongoing inflammation, but the status of soluble IL-6 receptor (sIL-6R) in these patients is unknown. We hypothesised that sIL-6R may be an important potentiator of IL-6 activity in CF associated lung disease. IL-6, sIL-6R and sgp130 (a natural antagonist of responses mediated by the sIL-6R) were analysed by ELISA and Western blot in bronchoalveolar lavage fluid (BALF) from 28 paediatric CF patients and nine non-CF controls. Total cell counts in CF were four fold higher compared to controls (median: 1.4 × 10⁶ cells/ml v. 0.35 × 10⁶ cells/ml in controls) (p < 0.001) and the infiltrate was dominated by neutrophils which were elevated by 89 fold (0.62 × 10⁶ cells/ml v. 0.007 × 10⁶ cells/ml in controls) (p < 0.001). Other markers of inflammation such as IL-8 and MCP-1 were elevated 17.5 and 3.8 fold respectively (IL-8; median: 1122 pg/ml v. 64 pg/ml in controls, p < 0.01 and MCP-1; median: 692 pg/ml v. 182 pg/ml in controls, p < 0.05). IL-6, although present in 23/32 CF BALF specimens compared to 1/9 controls (p < 0.01), was weakly expressed (median: 50 pg/ml). Expression of sIL-6R and sgp130 in CF was no different to control patients. We tested whether weak expression of all three molecules was due to degradation by CF BALF. Degradative activity was observed in association with BALF elastase activity and could be specifically blocked by serine protease inhibitors. Degradation of sIL-6R by purified serine proteases (elastase, cathepsin G and proteinase 3) was also observed leading to a loss of trans-signalling activity. Interestingly, sIL-6R was protected from proteolysis by interaction with IL-6. Our data identify and define a novel protease mediated deficiency of IL-6 signalling in the CF lung.