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.

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.

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 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.

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.

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.

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.

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.

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.

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.

The Brain Donation Program in South Korea.

PURPOSE: Obtaining brain tissue is critical to definite diagnosis and to furthering understanding of neurodegenerative diseases. The present authors have maintained the National Neuropathology Reference and Diagnostic Laboratories for Dementia in South Korea since 2016. We have built a nationwide brain bank network and are collecting brain tissues from patients with neurodegenerative diseases. We are aiming to facilitate analyses of clinic-pathological and image-pathological correlations of neurodegenerative disease and to broaden understanding thereof. MATERIALS AND METHODS: We recruited participants through two routes: from memory clinics and the community. As a baseline evaluation, clinical interviews, a neurological examination, laboratory tests, neuropsychological tests, and MRI were undertaken. Some patients also underwent amyloid PET. RESULTS: We recruited 105 participants, 70 from clinics and 35 from the community. Among them, 11 died and were autopsied. The clinical diagnoses of the autopsied patients included four with Alzheimer's disease (AD), two with subcortical vascular dementia, two with non-fluent variant primary progressive aphasia, one with leukoencephalopathy, one with frontotemporal dementia (FTD), and one with Creutzfeldt-Jakob disease (CJD). Five patients underwent amyloid PET: two with AD, one with mixed dementia, one with FTD, and one with CJD. CONCLUSION: The clinical and neuropathological information to be obtained from this cohort in the future will provide a deeper understanding of the neuropathological mechanisms of cognitive impairment in Asia, especially Korea.

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.

Coronin 1B serine 2 phosphorylation by p38α is critical for vascular endothelial growth factor-induced migration of human umbilical vein endothelial cells.

Coronin 1B is an actin-binding protein that regulates various cellular processes including cell motility. However, the role of coronin 1B in vascular cell migration remains controversial. Here, we examined the function of coronin 1B in vascular endothelial growth factor (VEGF)-induced migration of human umbilical vein endothelial cells (HUVECs) and investigated the mechanism by which coronin 1B regulates this cellular process. We found that depletion of coronin 1B increased the VEGF-induced migration of HUVECs. VEGF phosphorylated coronin 1B at Ser2 and stimulated its translocation to the leading edge of stimulated cells. Lentivirus-mediated overexpression of wild-type coronin 1B or a phosphodeficient coronin 1B S2A mutant decreased VEGF-induced transwell migration of HUVECs. Treatment with the p38 inhibitor SB203580 or depletion of p38α by small interfering RNA transfection decreased VEGF-induced coronin 1B phosphorylation. In vitro binding and kinase assays revealed that active p38α directly binds to and phosphorylates coronin 1B at Ser2. In addition, VEGF induced active p38α binding to coronin 1B in HUVECs. VEGF disrupted the interaction between coronin 1B and the actin-related protein (Arp)2/3 complex and p38α depletion prevented this VEGF-induced dissociation. These findings suggest that coronin 1B plays an inhibitory role in VEGF-induced migration of HUVECs and that VEGF-activated p38α phosphorylates coronin 1B at Ser2 and activates the Arp2/3 complex by liberating it from coronin 1B.

CaMKII-dependent myofilament Ca2+ desensitization contributes to the frequency-dependent acceleration of relaxation.

BACKGROUND: Previous studies suggest that CaMKII activity is required for frequency-dependent acceleration of relaxation (FDAR) in ventricular myocytes. We propose that the underlying mechanism involves CaMKII-dependent regulation of myofilament Ca(2+) sensitivity. METHODS AND RESULTS: Cardiac function was measured in mice using murine echo machine. [Ca(2+)]i and sarcomere length were measured by IonOptix Ca(2+) image system. Increasing pacing rate from 0.5 to 4 Hz in left ventricular myocytes induced frequency-dependent myofilament Ca(2+) desensitization (FDMCD) and FDAR. Acute inhibition of PKA or PKC had no effect, whereas CaMKII inhibition abolished both FDMCD and FDAR. Co-immunoprecipitation of CaMKII and troponin I (TnI) has been detected and CaMKII inhibition significantly reduced serine residue phosphorylation of TnI. Finally, chronic inhibition of CaMKII in vivo reduced TnI phosphorylation and abolished both FDAR and FDMCD, leading to impaired diastolic function. CONCLUSIONS: Our results suggest that CaMKII-dependent TnI phosphorylation is involved in FDMCD and the consequent FDAR and that CaMKII inhibition removes this mechanism and thus induces diastolic dysfunction.

Coronin 1A depletion protects endothelial cells from TNFα-induced apoptosis by modulating p38ß expression and activation.

Coronins are conserved actin-binding proteins that regulate various cellular processes such as migration and endocytosis. Among coronin family members, coronin 1A is highly expressed in hematopoietic lineage cells where it regulates cell homeostasis. However, the expression and function of coronin 1A in endothelial cells have not yet been elucidated. We found that coronin 1A is expressed in the human umbilical vein endothelial cell (HUVEC) and human brain microvascular endothelial cell (HBMVEC). In HUVEC depleted of coronin 1A by siRNA transfection, tumor necrosis factor α (TNFα)+cyclohexamide (CHX) treatment resulted in a decrease in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive apoptotic cells. Coronin 1A depletion also resulted in the suppression of caspase 3 and poly(ADP-ribose) polymerase cleavage and a reduction in caspase 3 activity. Next, we examined TNFα-induced activation of several pro- and anti-apoptotic signaling molecules to find the target molecule of coronin 1A and found that p38 phosphorylation was enhanced by TNFα stimulation in coronin 1A-depleted HUVEC. Among the p38 isoforms, the expression of p38ß was significantly upregulated after coronin 1A depletion, suggesting that the expression and phosphorylation of anti-apoptotic p38ß were significantly induced in coronin 1A-depleted HUVEC. Inhibition of p38ß upregulation in coronin 1A-depleted HUVEC restored the cleavage of caspase 8 and caspase 3 and induced more apoptosis than in coronin 1A-depleted HUVEC in response to TNFα+CHX. These findings suggest that coronin 1A modulates endothelial cell apoptosis by regulating p38ß expression and activation.

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.

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.

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.

HB-EGF induces cardiomyocyte hypertrophy via an ERK5-MEF2A-COX2 signaling pathway.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family that binds to and activates the EGF receptor. Transactivated by angiotensin II, ET-1, and various growth factors in cardiomyocytes, HB-EGF is known to induce cardiac hypertrophy via the PI3K-Akt, MAP kinase, and JAK-STAT pathways. However, little is known about the potential involvement of the ERK5 pathway in HB-EGF-induced cardiac hypertrophy. In the present report, we identify and characterize a novel MEK5-ERK5 pathway that is involved in HB-EGF-induced cardiomyocyte hypertrophy. HB-EGF (10ng/ml) significantly increased [(3)H]-leucine incorporation and atrial natriuretic factor (ANF) mRNA expression in H9c2 cells. In addition, HB-EGF activated a MEK5-ERK5 pathway. Pretreatment with the EGFR inhibitor AG1478 attenuated the activation of ERK5. Blockade of MEK5-ERK5 signaling using MEK5 siRNA reduced the ability of HB-EGF to increase cell size and the expression of ANF mRNA, suggesting the involvement of an EGFR-ERK5 pathway in HB-EGF-induced cardiomyocyte hypertrophy. We further analyzed cyclooxygenase-2 (COX-2). HB-EGF enhanced the expression of COX-2, a response mediated by MEK5-ERK5 signaling, while the COX-2 inhibitor rofecoxib attenuated HB-EGF-induced ANF mRNA expression, suggesting that COX-2 is also associated with HB-EGF-induced cardiomyocyte hypertrophy. It has been known that ERK5 activates the myocyte enhancer factor (MEF) 2 family of transcription factor, we next tested whether activation of MEF2A contributes to HB-EGF-induced COX-2 expression. Inhibition of MEF2A using siRNA attenuated HB-EGF-induced COX-2, ANF expression and cell size. In conclusion, HB-EGF induces cardiomyocyte hypertrophy through an EGFR-ERK5-MEF2A-COX-2 pathway. Our findings will help us to better understand the molecular mechanisms behind HB-EGF-induced cardiomyocyte hypertrophy.