Diesel exhaust particle exposure exacerbates ciliary and epithelial barrier dysfunction in the multiciliated bronchial epithelium models.

Airway epithelium, the first defense barrier of the respiratory system, facilitates mucociliary clearance against inflammatory stimuli, such as pathogens and particulates inhaled into the airway and lung. Inhaled particulate matter 2.5 (PM2.5) can penetrate the alveolar region of the lung, and it can develop and exacerbate respiratory diseases. Although the pathophysiological effects of PM2.5 in the respiratory system are well known, its impact on mucociliary clearance of airway epithelium has yet to be clearly defined. In this study, we used two different 3D in vitro airway models, namely the EpiAirway-full-thickness (FT) model and a normal human bronchial epithelial cell (NHBE)-based air-liquid interface (ALI) system, to investigate the effect of diesel exhaust particles (DEPs) belonging to PM2.5 on mucociliary clearance. RNA-sequencing (RNA-Seq) analyses of EpiAirway-FT exposed to DEPs indicated that DEP-induced differentially expressed genes (DEGs) are related to ciliary and microtubule function and inflammatory-related pathways. The exposure to DEPs significantly decreased the number of ciliated cells and shortened ciliary length. It reduced the expression of cilium-related genes such as acetylated α-tubulin, ARL13B, DNAH5, and DNAL1 in the NHBEs cultured in the ALI system. Furthermore, DEPs significantly increased the expression of MUC5AC, whereas they decreased the expression of epithelial junction proteins, namely, ZO1, Occludin, and E-cadherin. Impairment of mucociliary clearance by DEPs significantly improved the release of epithelial-derived inflammatory and fibrotic mediators such as IL-1ß, IL-6, IL-8, GM-CSF, MMP-1, VEGF, and S100A9. Taken together, it can be speculated that DEPs can cause ciliary dysfunction, hyperplasia of goblet cells, and the disruption of the epithelial barrier, resulting in the hyperproduction of lung injury mediators. Our data strongly suggest that PM2.5 exposure is directly associated with ciliary and epithelial barrier dysfunction and may exacerbate lung injury.

Coronin 1B regulates the TNFα-induced apoptosis of HUVECs by mediating the interaction between TRADD and FADD.

Coronin 1B is an actin-binding protein that plays important roles in actin-dependent cellular processes. We previously reported that coronin 1B is involved in vascular endothelial cell growth factor-induced migration of human umbilical vein endothelial cells (HUVECs). However, the role of coronin 1B in tumor necrosis factor alpha (TNFα)-induced endothelial cell apoptosis remained unknown. In this study, we investigated whether coronin 1B affects TNFα-induced HUVEC apoptosis and sought to elucidate the mechanism by which coronin 1B regulates this cellular process. Depletion of coronin 1B by siRNA transfection decreased TNFα-induced apoptosis of HUVECs, as determined by MTT, terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase-3 activity assays. Coronin 1B depletion also decreased caspase-8 cleavage via a JNK-independent pathway. Coronin 1B interacted with Fas-associated death domain protein (FADD) in both a plasmid overexpression system in HEK293T cells and at the endogenous protein level in TNFα-stimulated HUVECs. Immunoprecipitation and in situ proximity ligation assays showed that coronin 1B depletion diminished the interaction between TNFα-induced TNF receptor-1-associated death domain protein (TRADD) and FADD, suggesting that coronin 1B is required for the TNFα-induced TRADD and FADD interaction and subsequent caspase-8/caspase-3 cascade activation, ultimately leading to apoptosis.

Binding of coronin 1B to TßRI negatively regulates the TGFß1 signaling pathway.

Coronin 1B is an actin-binding protein that regulates several actin-dependent cellular processes including migration and endocytosis. However, the role of coronin 1B in the tumor growth factor (TGF)ß signaling pathway is largely unknown. Here, we investigated whether coronin 1B affects the TGFß signaling cascade and found that coronin 1B negatively regulates the TGFß signaling pathway. Immunoprecipitation and glutathione-S-transferase-pulldown assays revealed that coronin 1B directly associated with TGFß receptor I (TßRI). Overexpression of coronin 1B inhibited the TGFß1-induced interaction between TßRI and Smad2/3 in plasmid-transfected HEK293T cells. Coronin 1B was basally bound to TßRI in vascular smooth muscle cells (VSMCs), but TGFß1 stimulation did not affect their association, suggesting constitutive binding between coronin 1B and TßRI. Overexpression of coronin 1B suppressed TGFß1-induced activation of a Smad-binding element-luciferase reporter construct and a plasminogen activator inhibitor (PAI)-1 promoter-luciferase reporter construct in HEK293T cells. By contrast, depletion of coronin 1B by siRNA transfection increased TGFß1-induced Smad2/3 phosphorylation and PAI-1 expression in VSMCs. These results suggest that coronin 1B regulates the TGFß1 signaling cascade by constitutively interacting with TßRI and inhibiting the binding of Smad2/3 to TßRI in response to TGFß1 stimulation.

Stretch current-induced abnormal impulses in CaMKIIδ knockout mouse ventricular myocytes.

BACKGROUND: CaMKII activation is proarrhythmic in heart failure where myocardium is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle has not been well understood. OBJECTIVE: We tested abnormal impulse inducibility by stretch current in myocytes isolated from CaMKIIδ knockout (KO) mouse left ventricle (LV) where CaMKII activity is reduced by ≈ 62%. METHODS AND RESULTS: Action potentials were recorded by whole-cell patch clamp, and abnormal impulses were induced in LV myocytes by a simulation of stretch-activated channel (SAC) current. SAC activation failed to induce abnormal impulses in wild type (WT) myocytes but steadily produced early after-depolarizations and automaticity in KO myocytes in which an increase in L-type calcium channel (LTCC) current (I(Ca)) and a reduction of sarcoplasmic reticulum Ca(2+) leak and action potential duration (APD) were observed. The abnormal impulses were not suppressed by CaMKII inhibitor AIP whereas a low concentration of nifedipine eliminated abnormal impulses without shortening APD, implicating I(Ca) in promoting stretch-induced abnormal impulses. In addition, APD prolongation by LTCC opener S(-)Bay K 8644 or isoproterenol facilitated abnormal impulse induction in WT ventricular myocytes even in the presence of CaMKII inhibitor AIP, whereas APD prolongation by K(+) channel blocker 4-aminopyridine promoted abnormal impulses in KO myocytes but not in WT myocytes. CONCLUSION: I(Ca) activation plays a central role in stretch-induced abnormal impulses and APD prolongation is arrhythmogenic only when I(Ca) is highly activated. At increased I(Ca) activation, CaMKII inhibition cannot suppress abnormal impulse induction.

Exogenous 8-Hydroxydeoxyguanosine Attenuates PM2.5-Induced Inflammation in Human Bronchial Epithelial Cells by Decreasing NLRP3 Inflammasome Activation.

Particulate matter 2.5 (PM2.5) induces lung injury by increasing the generation of reactive oxygen species (ROS) and inflammation. ROS aggravates NLRP3 inflammasome activation, which activates caspase-1, IL-1ß, and IL-18 and induces pyroptosis; these factors propagate inflammation. In contrast, treatment with exogenous 8-hydroxydeoxyguanosine (8-OHdG) decreases RAC1 activity and eventually decreases dinucleotide phosphate oxidase (NOX) and ROS generation. To establish modalities that would mitigate PM2.5-induced lung injury, we evaluated whether 8-OHdG decreased PM2.5-induced ROS generation and NLRP3 inflammasome activation in BEAS-2B cells. CCK-8 and lactate dehydrogenase assays were used to determine the treatment concentration. Fluorescence intensity, Western blotting, enzyme-linked immunosorbent assay, and immunoblotting assays were also performed. Treatment with 80 µg/mL PM2.5 increased ROS generation, RAC1 activity, NOX1 expression, NLRP3 inflammasome (NLRP3, ASC, and caspase-1) activity, and IL-1ß and IL-18 levels in cells; treatment with 10 µg/mL 8-OHdG significantly attenuated these effects. Furthermore, similar results, such as reduced expression of NOX1, NLRP3, ASC, and caspase-1, were observed in PM2.5-treated BEAS-2B cells when treated with an RAC1 inhibitor. These results show that 8-OHdG mitigates ROS generation and NLRP3 inflammation by inhibiting RAC1 activity and NOX1 expression in respiratory cells exposed to PM2.5.

The PPARδ ligand L-165041 inhibits VEGF-induced angiogenesis, but the antiangiogenic effect is not related to PPARδ.

Peroxisome proliferator-activated receptor (PPAR)δ is known to be expressed ubiquitously and involved in lipid and glucose metabolism. Recent studies have demonstrated that PPARδ is expressed in endothelial cells (ECs) and plays a potential role in endothelial survival and proliferation. Although PPARα and PPARγ are well recognized to play anti-inflammatory, antiproliferative, and antiangiogenic roles in ECs, the general effect of PPARδ on angiogenesis in ECs remains unclear. Thus, we investigated the effect of the PPARδ ligand L-165041 on vascular EC proliferation and angiogenesis in vitro as well as in vivo. Our data show that L-165041 inhibited VEGF-induced cell proliferation and migration in human umbilical vein ECs (HUVECs). L-165041 also inhibited angiogenesis in the Matrigel plug assay and aortic ring assay. Flow cytometric analysis indicated that L-165041 reduced the number of ECs in the S phase and the expression levels of cell cycle regulatory proteins such as cyclin A, cyclin E, CDK2, and CDK4; phosphorylation of the retinoblastoma protein was suppressed by pretreatment with L-165041. We confirmed whether these antiangiogenic effects of L-165041 were PPARδ-dependent using GW501516 and PPARδ siRNA. GW501516 treatment did not inhibit VEGF-induced angiogenesis, and transfection of PPARδ siRNA did not reverse this antiangiogenic effect of L-165041, suggesting that the antiangiogenic effect of L-165041 on ECs is PPARδ-independent. Together, these data indicate that the PPARδ ligand L-165041 inhibits VEGF-stimulated angiogenesis by suppressing the cell cycle progression independently of PPARδ. This study highlights the therapeutic potential of L-165041 in the treatment of many disorders related to pathological angiogenesis.

CaMKII inhibition in heart failure, beneficial, harmful, or both.

Calmodulin-dependent protein kinase II (CaMKII) has been proposed to be a therapeutic target for heart failure (HF). However, the cardiac effect of chronic CaMKII inhibition in HF has not been well understood. We have tested alterations of Ca(2+) handling, excitation-contraction coupling, and in vivo ß-adrenergic regulation in pressure-overload HF mice with CaMKIIδ knockout (KO). HF was produced in wild-type (WT) and KO mice 1 wk after severe thoracic aortic banding (sTAB) with a continuous left ventricle (LV) dilation and reduction of ejection fraction for up to 3 wk postbanding. Cardiac hypertrophy was similar between WT HF and KO HF mice. However, KO HF mice manifested exacerbation of diastolic function and reduction in cardiac reserve to ß-adrenergic stimulation. Compared with WT HF, L-type calcium channel current (I(Ca)) density in KO HF LV was decreased without changes in I(Ca) activation and inactivation kinetics, whereas I(Ca) recovery from inactivation was accelerated and Ca(2+)-dependent I(Ca) facilitation, a positive staircase blunted in WT HF, was recovered. However, I(Ca) response to isoproterenol was reduced. KO HF myocytes manifested dramatic decrease in sarcoplasmic reticulum (SR) Ca(2+) leak and slowed cytostolic Ca(2+) concentration decline. Sarcomere shortening was increased, but relaxation was slowed. In addition, an increase in myofilament sensitivity to Ca(2+) and the slow skeletal muscle troponin I-to-cardiac troponin I ratio and interstitial fibrosis and a decrease in Na/Ca exchange function and myocyte apoptosis were observed in KO HF LV. CaMKIIδ KO cannot suppress severe pressure-overload-induced HF. Although cellular contractility is improved, it reduces in vivo cardiac reserve to ß-adrenergic regulation and deteriorates diastolic function. Our findings challenge the strategy of CaMKII inhibition in HF.

Alterations of L-type calcium current and cardiac function in CaMKII{delta} knockout mice.

RATIONALE: Recent studies have highlighted important roles of CaMKII in regulating Ca(2+) handling and excitation-contraction coupling. However, the cardiac effect of chronic CaMKII inhibition has not been well understood. OBJECTIVE: We have tested the alterations of L-type calcium current (I(Ca)) and cardiac function in CaMKIIdelta knockout (KO) mouse left ventricle (LV). METHODS AND RESULTS: We used the patch-clamp method to record I(Ca) in ventricular myocytes and found that in KO LV, basal I(Ca) was significantly increased without changing the transmural gradient of I(Ca) distribution. Substitution of Ba(2+) for Ca(2+) showed similar increase in I(Ba). There was no change in the voltage dependence of I(Ca) activation and inactivation. I(Ca) recovery from inactivation, however, was significantly slowed. In KO LV, the Ca(2+)-dependent I(Ca) facilitation (CDF) and I(Ca) response to isoproterenol (ISO) were significantly reduced. However, ISO response was reversed by beta2-adrenergic receptor (AR) inhibition. Western blots showed a decrease in beta1-AR and an increase in Ca(v)1.2, beta2-AR, and Galphai3 protein levels. Ca(2+) transient and sarcomere shortening in KO myocytes were unchanged at 1-Hz but reduced at 3-Hz stimulation. Echocardiography in conscious mice revealed an increased basal contractility in KO mice. However, cardiac reserve to work load and beta-adrenergic stimulation was reduced. Surprisingly, KO mice showed a reduced heart rate in response to work load or beta-adrenergic stimulation. CONCLUSIONS: Our results implicate physiological CaMKII activity in maintaining normal I(Ca), Ca(2+) handling, excitation-contraction coupling, and the in vivo heart function in response to cardiac stress.

Dynamic Kv4.3-CaMKII unit in heart: an intrinsic negative regulator for CaMKII activation.

AIMS: Reduction of transient outward current (I(to)) and excessive activation of Ca(2+)/Calmodulin-dependent kinase II (CaMKII) are general features of ventricular myocytes in heart failure. We hypothesize that alterations of I(to) directly regulate CaMKII activation in cardiomyocytes. METHODS AND RESULTS: A dynamic coupling of I(to) channel subunit Kv4.3 and inactive CaMKII was discovered in cardiomyocytes with the membrane predominant distribution by co-immunoprecipitation and fluorescence resonance energy transfer techniques. CaMKII dissociation from Kv4.3-CaMKII units caused a significant increase in CaMKII autophosphorylation and L-type calcium current (I(Ca)) facilitation. I(Ca) facilitation was blunted by the compartmental Ca²(+) chelator BAPTA but unaffected by bulk Ca²(+) chelator EGTA, implicating membrane-localized CaMKII. Kv4.3 overexpression reduced basal CaMKII autophosphorylation in myocytes and eliminated Ca²(+)-induced CaMKII activation. Kv4.3 blocks CaMKII activation by binding to the calmodulin binding sites, whereas Kv4.3 uncoupling releases these sites and leads to a substantial CaMKII activation. CONCLUSION: Our results uncovered an important mechanism that regulates CaMKII activation in the heart and implicate I(to) channel alteration in pathological CaMKII activation.

Publication trends in South Korean research on particulate matter and health effects during two decades (2000-2019).

Ambient particulate matter is a serious risk factor for health outcomes associated with various diseases, including respiratory and cardiovascular diseases. South Korea is one of the Organization for Economic Cooperation and Development (OECD) countries with the highest concentration of ambient particulate matter. The purpose of this study is to identify the status of research on particulate matter and associated health effects in South Korea through bibliometric methods. Scientific articles related to particulate matter (PM10 and PM2.5) and their effects on health published during the last two decades (2000-2019) were retrieved using the Scopus database. The total number of publications on PM10 and health effects was 518, and 197 publications were authored on PM2.5 and health effects. This number has increased substantially in the last 3 years. The institution and the country that contributed the highest number of publications to ambient particulate matter research were the Seoul National University and the United States, respectively. Publications on the effects of ambient particulates on children, the elderly, or pregnant women accounted for less than 30% of all retrieved publications. Publications on nitrogen oxides (NOx), sulfur oxide (SO2), or polycyclic aromatic hydrocarbons (PAHs) accounted for approximately 30% and 20% of health effects-associated publications retrieved from Scopus concerning PM10 and PM2.5 research, respectively. Analysis of author keywords showed that mortality, respiratory diseases, cardiovascular disease, and oxidative stress were main research topics on particulate matter and health effects. Our study provides information that can be used to grasp research trends and not covered research topics on health effects of particulate matter in Korea.

Positive Effect of Air Purifier Intervention on Baroreflex Sensitivity and Biomarkers of Oxidative Stress in Patients with Coronary Artery Disease: A Randomized Crossover Intervention Trial.

Exposure to fine particulate matter increases the risk of cardiovascular morbidity and mortality. Few studies have tested the beneficial effect of indoor air filtration intervention in patients with cardiovascular disease. The aim of this study is to investigate the effect of air filtration on mitigating cardiovascular health in patients with coronary artery disease. This randomized, double-blind, crossover study is conducted with 38 coronary artery disease patients. The intervention consists of the following three periods: two-week active and sham air filtration interventions, with a two-week washout period. The indoor PM2.5 concentration is continuously monitored during the entire study period. We measure the blood pressure, heart rate variability, baroreflex sensitivity, autonomic function test results, and endothelial function. The two-week active air filtration intervention for two weeks reduces the average indoor concentration of PM2.5 by 33.9%. The indoor PM2.5 concentration is significantly correlated to cross-correlation baroreflex sensitivity. Active air filtration is significantly associated with a decrease in the indicator of oxidative stress represented as 8-hydroxy-2'-deoxyguanosine. This study shows that a short-term air filtration intervention improved baroreflex sensitivity and might reduce oxidative stress in coronary artery disease patients. These findings suggest that the use of an air purifier could mitigate the recurrence of cardiovascular disease events in patients with coronary artery disease.

Publication trends in research on particulate matter and health impact over a 10-year period: 2009-2018.

Exposure to ambient particulate matter is a major health risk factor for numerous diseases, including those of the cardiovascular and respiratory varieties. The aim of this study was to estimate the latest global research activities regarding particulate matter and health impact. We performed a bibliometric analysis of this field's scientific publication trends over a decade (2009-2018). Publications were retrieved from the Scopus and Web of Science databases using the search terms "particulate matter," "fine particulate matter," "health impact," and their synonyms. The literature on health impact in the research fields of particulate matter (PM10) and fine particulate matter (PM2.5) trended to significantly increase over the decade in consideration. It appears to have been led by researchers of the United States and China. Worldwide research on particulate matter and health effects has focused primarily on respiratory and cardiovascular diseases. The precursors to and components of particulate matter (such as nitrogen dioxide, polycyclic aromatic hydrocarbon, sulfur dioxide, and black carbon) were also popular research topics in this field. Research on children, older adults, and pregnant women, who are most vulnerable to the health effects of air pollution, has increased dramatically over the past 10 years. Our findings provide the information necessary to predict unmet research topics and future research needs.

Age and Gender Effects on Genotoxicity in Diesel Exhaust Particles Exposed C57BL/6 Mice.

There is growing evidence that the accumulation of DNA damage induced by fine particulate matter (PM2.5) exposure is an underlying mechanism of pulmonary disease onset and progression. However, there is a lack of experimental evidence on whether common factors (age, gender) affect PM2.5 induced genomic damage. Here, we assessed the DNA damage potency of PM2.5 using conventional genotoxicity testing in old male and female mice aged 8 and 40 weeks. Mice were intratracheally instilled with diesel exhaust PM2.5 (DEP, NIST SRM 1650b), twice a week for 4 weeks. Exposure to DEP was not associated with an increase in the frequency of micronucleated polychromatic erythrocytes and did not induce a systemic genotoxic effect in the bone marrow. Meanwhile, the results from the comet assay showed a significant increase in DNA damage in DEP exposed mouse lung specimens. The positive relationship between DEP exposure and DNA damage is stronger in the older than in the younger group. Statistical analysis showed that there was a modifying effect of age on the association between PM2.5 exposure and DNA damage. Our results suggest that the age factor should be considered to better understand the cellular adverse effects of PM2.5.

Determination of Genotoxicity Attributed to Diesel Exhaust Particles in Normal Human Embryonic Lung Cell (WI-38) Line.

Several epidemiological studies concluded that inhalation of diesel exhaust particles (DEP) is associated with an increase in the relative risk of lung cancer. In vitro research evaluating the genetic damage and/or changes in gene expression have been attempted to explain the relationship between DEP exposure and carcinogenicity. However, to date, investigations have been largely confined to studies in immortalized or tumorigenic epithelial cell models. Few studies have investigated damage at the chromosomal level to DEP exposure in normal cell lines. Here, we present the genotoxic effects of DEP in normal cells (embryonic human lung fibroblasts) by conventional genotoxicity testing (micronuclei (MN) and comet assay). We show the differentially expressed genes and enriched pathways in DEP-exposed WI-38 cells using RNA sequencing data. We observed a significant increase in single-strand DNA breaks and the frequency of MN in DEP-exposed cells in a dose-dependent manner. The differentially expressed genes following DEP exposure were significantly enriched in the pathway for responding to xenobiotics and DNA damage. Taken together, these results show that DEP exposure induced DNA damage at the chromosomal level in normal human lung cells and provide information on the expression of genes associated with genotoxic stress.

PPARdelta activation inhibits angiotensin II induced cardiomyocyte hypertrophy by suppressing intracellular Ca2+ signaling pathway.

Peroxisome proliferator-activated receptors delta (PPARdelta) is known to be expressed ubiquitously, and the predominant PPAR subtype of cardiac cells. However, relatively less is known regarding the role of PPARdelta in cardiac cells except that PPARdelta ligand treatment protects cardiac hypertrophy by inhibiting NF-kappaB activation. Thus, in the present study, we examined the effect of selective PPARdelta ligand L-165041 on angiotensin II (AngII) induced cardiac hypertrophy and its underlying mechanism using cardiomyocyte. According to our data, L-165041 (10 microM) inhibited AngII-induced [(3)H] leucine incorporation, induction of the fetal gene atrial natriuretic factor (ANF) and increase of cardiomyocyte size. Previous studies have implicated the activation of focal adhesion kinase (FAK) in the progress of cardiomyocyte hypertrophy. L-165041 pretreatment significantly inhibited AngII-induced intracellular Ca(2+) increase and subsequent phosphorylation of FAK. Further experiment using Ca(2+) ionophore A23187 confirmed that Ca(2+) induced FAK phosphorylation, and this was also blocked by L-165041 pretreatment. In addition, overexpression of PPARdelta using adenovirus significantly inhibited AngII-induced intracellular Ca(2+) increase and FAK expression, while PPARdelta siRNA treatment abolished the effect of L-165041. These data indicate that PPARdelta ligand L-165041 inhibits AngII induced cardiac hypertrophy by suppressing intracellular Ca(2+)/FAK/ERK signaling pathway in a PPARdelta dependent mechanism.

PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways.

Myocyte apoptosis plays an important role in myocardial infarction and cAMP is crucial in the regulation of myocyte apoptosis. Phosphodiesterase-4 (PDE4) inhibitor blocks the hydrolysis of cAMP via inhibition of PDE4 and is attractive candidate for novel anti-inflammatory drugs. However, its function in cardiovascular diseases and cardiomyocyte apoptosis is unclear. Therefore, we investigated whether roflumilast, a PDE4 inhibitor, exerts protective effect against NO-induced apoptosis in both of H9c2 cells and neonatal rat cardiomyocytes (NRCMs), focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). According to our data, intracellular cAMP was increased by roflumilast treatment in H9c2 cells and NRCMs. Roflumilast inhibited SNP-induced apoptosis and this effect was reversed by PKA specific inhibitor H-89 and KT-5720. In addition, PKA specific activator N(6)-benzoyladenosine 3',5-cyclic monophosphate (N(6)Bz-cAMP) mimicked the effects of roflumilast. CREB phosphorylation by roflumilast was also inhibited by H-89, indicating that roflumilast protects SNP-induced apoptosis via PKA-dependent pathway. Roflumilast increased Epac1/GTP-Rap1 and the protective effect was abolished by Epac1 siRNA transfection, demonstrating that Epac signaling was also involved in this protective response. In support, Epac specific activator 8-(4-chlrorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) protected SNP-induced apoptosis. PI3K/Akt inhibitor LY294002 blocked roflumilast-induced Akt phosphorylation and protective effect. Furthermore, inhibition of Epac1 with siRNA had no effect on roflumilast-induced CREB phosphorylation, whereas inhibited Akt phosphorylation, implicating that Akt phosphorylation was regulated by Epac pathway. In addition, it was also observed that rolipram and cilomilast exert similar effects as roflumilast. In summary, our data indicate that roflumilast protects NO-induced apoptosis via both cAMP-PKA/CREB and Epac/Akt-dependent pathway. Our study suggests a possibility of PDE4 inhibitor roflumilast as a potential therapeutic agent against myocardial ischemia/reperfusion (I/R) injury.

Zileuton, a 5-Lipoxygenase Inhibitor, Exerts Anti-Angiogenic Effect by Inducing Apoptosis of HUVEC via BK Channel Activation.

The arachidonic acid metabolism through 5-lipoxygenase (5-LO) pathways is involved in modulating both tumorigenesis and angiogenesis. Although anti-carcinogenic activities of certain 5-LO inhibitors have been reported, the role of zileuton, a well known 5-LO inhibitor, on the endothelial cell proliferation and angiogenesis has not been fully elucidated. Here, we report that zileuton has an anti-angiogenic effect, and the underlying mechanisms involved activation of the large-conductance Ca2+-activated K+ (BK) channel. Our results show that zileuton significantly prevented vascular endothelial growth factor (VEGF)-induced proliferation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as in vivo. However, such anti-angiogenic effect of zileuton was abolished by iberiotoxin (IBTX), a BK channel blocker, suggesting zileuton-induced activation of BK channel was critical for the observed anti-angiogenic effect of zileuton. Furthermore, the anti-angiogenic effect of zileuton was, at least, due to the activation of pro-apoptotic signaling cascades which was also abolished by IBTX. Additionally, zileuton suppressed the expression of VCAM-1, ICAM-1, ETS related gene (Erg) and the production of nitric oxide (NO). Taken together, our results show that zileuton prevents angiogenesis by activating the BK channel dependent-apoptotic pathway, thus highlighting its therapeutic capacity in angiogenesis-related diseases, such as cancer.

Cigarette smoke extracts and cadmium induce COX-2 expression through γ-secretase-mediated p38 MAPK activation in C6 astroglia cells.

Exposure to cigarette smoke has been implicated in the progression of cerebrovascular and neurological disorders like stroke through inflammation and blood-brain barrier disruption. In this study, we investigated the signaling cascade activated by cigarette smoke extracts (CSE) and cadmium (Cd) resulting in the COX-2 induction in C6 rat astroglia cells. CSE or Cd induced Notch1 cleavage and activated p38 MAPK and CREB signaling pathways in C6 astroglia cells. Knockdown of nicastrin using siRNA or γ-secretase inhibitors, DAPT and L-685,486, reduced Notch1 cleavage and phosphorylation of p38 MAPK and CREB, while phosphorylation of ERK and JNK remained unaffected. Additionally, the blockage of γ-secretase activity did not show any effect on the phosphorylation of AKT, another upstream activator of CREB, indicating that γ-secretase-mediated CREB activation occurs via p38 MAPK. γ-secretase inhibitor also inhibited the CSE and Cd-mediated increase in the expression of COX-2. Furthermore, recombinant overexpression of Notch1 intracellular domain resulted in an increase in the expression of COX-2. Notch signaling induced by CSE and Cd induced apoptosis in C6 cells. Our results demonstrate that CSE exposure activated the p38 MAPK and CREB-mediated induction in COX-2 expression in astrocytes via γ-secretase-mediated Notch1 signaling. Our data provides novel insights into the potential mechanism of pro-inflammatory response activated by exposure to cigarette smoke.

Plasma ATG5 is increased in Alzheimer's disease.

Alzheimer's disease (AD) is a major cause of dementia. Growing evidence suggests that dysregulation of autophagy, a cellular mechanism essential for self-digestion of damaged proteins and organelles, is involved in neurological degenerative diseases including AD. Previously, we reported that autophagosomes are increased in the brains of AD mouse model. However, the plasma levels of autophagic markers have not yet been investigated in patients with AD. In this study, we investigated the expression of autophagy-related genes 5 and 12 (ATG5 and ATG12, respectively) in cells in vitro upon amyloid-beta (Aß) treatment and in the plasma of AD patients. ATG5-ATG12 complex levels were increased in primary rat cortical neurons and human umbilical vein endothelial cells after Aß treatment. Furthermore, we compared plasma from 69 patients with dementia, 82 patients with mild cognitive impairment (MCI), and 127 cognitively normal control participants. Plasma levels of ATG5 were significantly elevated in patients with dementia (149.3 ± 7.5 ng/mL) or MCI (152.9 ± 6.9 ng/mL) compared with the control subjects (129.0 ± 4.1 ng/mL) (p = 0.034, p = 0.016, respectively). Our results indicate that alterations in the plasma ATG5 levels might be a potential biomarker in patients at risk for AD.

HB-EGF induces delayed STAT3 activation via NF-kappaB mediated IL-6 secretion in vascular smooth muscle cell.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family that binds to and activates EGF receptor, and is expressed in a variety of tissues, predominantly in the lung, heart, brain and skeletal muscle. HB-EGF is known to induce vascular smooth muscle cell (VSMC) proliferation by activating PI3K-Akt and MAPK pathway. However, our preliminary data showed that Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway was also involved in HB-EGF induced VSMC proliferation. More interestingly, HB-EGF (10 ng/ml) induced a biphasic activation of STAT3 (early at 5 min and late at 60-120 min). Therefore, we tried to elucidate the underlying mechanism of this delayed STAT3 activation by HB-EGF in VSMCs. First, we examined the effect of HB-EGF on interleukin-6 (IL-6) mRNA expressions, since IL-6 have been implicated in the regulation of STAT3 activation. According to our data, HB-EGF increased transcription of IL-6, cardiotrophin-1 (CT-1), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). The secretion of IL-6 was also increased by HB-EGF. Furthermore, these HB-EGF-mediated up-regulation of IL-6 mRNA expression and secretion were inhibited by NF-kappaB inhibitor Bay117082 (2.5 microM) treatment suggesting involvement of NF-kappaB pathway. Again, the late activation of STAT3 by HB-EGF was abolished by both Bay117082 and IL-6 neutralizing antibody (1 microg/ml) indicating IL-6 is a key molecule in the delayed activation of STAT3 by HB-EGF. In addition, IL-6 neutralizing antibody inhibited both HB-EGF conditioned media induced STAT3 activation and HB-EGF induced VSMC proliferation. In conclusion, IL-6 plays an important role in the delayed activation of STAT3 and VSMC proliferation induced by HB-EGF.