Yuh-Lin Wu

Resistin increases lipid accumulation by affecting class A scavenger receptor, CD36 and ATP-binding cassette transporter-A1 in macrophages

AIMS Resistin promotes macrophage-foam cell formation, but the mechanisms are unclear. In macrophages, lipid uptake is regulated by scavenger receptors (SR-A and CD36), while the cholesterol efflux is regulated by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. We investigated the mechanisms underlying the dysregulation by resistin of these regulators leading to promotion of lipid accumulation in bone marrow-derived macrophages. MAIN METHODS Western blotting, real-time PCR and oil red O staining were performed. KEY FINDINGS Resistin exacerbated lipid accumulation in oxLDL-treated macrophages. Resistin treatment of oxLDL-untreated macrophages showed increased SR-A and CD36 mRNA and protein levels, and decreased ABCA1 protein level, while having no effect on SR-BI or ABCG1 expression. Up-regulation of SR-A and CD36 by resistin resulted from activation of AP-1 and PPARgamma, respectively, and this was confirmed by the lack of activation of either after AP-1 inhibition using curcumin or SP600125, or PPARgamma inhibition using GW9662, respectively. The down-regulation of ABCA1 by resistin was not accompanied by a reduced mRNA level or an activation of LXRalpha/RXR, but resulted from enhanced protein degradation as revealed by the abolition of the down-regulation after inhibition of the proteasome pathway using ALLN or MG-132. A combined inhibition by SP600125, GW9662 and ALLN prevented resistin-induced exacerbation of lipid accumulation in oxLDL-treated macrophages. SIGNIFICANCE Resistin promotes foam cell formation via dysregulation of SR-A, CD36 and ABCA1. SR-A and CD36 are transcriptionally up-regulated by resistin through AP-1 and PPARgamma, respectively, whereas ABCA1 is down-regulated by resistin through proteasome-mediated enhancement of protein degradation.

EGb761 ameliorates the formation of foam cells by regulating the expression of SR-A and ABCA1: role of haem oxygenase-1

AIMS Accumulation of foam cells in the intima is a hallmark of early-stage atherosclerotic lesions. Ginkgo biloba extract (EGb761) has been reported to exert anti-oxidative and anti-inflammatory properties in atherosclerosis, yet the significance and the molecular mechanisms of action of EGb761 in the formation of macrophage foam cells are not fully understood. METHODS AND RESULTS Treatment with EGb761 resulted in a dose-dependent decrease in oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, a consequence that was due to a decrease in cholesterol uptake and an increase in cholesterol efflux. Additionally, EGb761 significantly down-regulated the mRNA and protein expression of class A scavenger receptor (SR-A) by decreasing expression of activator protein 1 (AP-1); however, EGb761 increased the protein stability of ATP-binding cassette transporter A1 (ABCA1) by reducing calpain activity without affecting ABCA1 mRNA expression. Small interfering RNA (siRNA) targeting haem oxygenase-1 (HO-1) abolished the EGb761-induced protective effects on the expression of AP-1, SR-A, ABCA1, and calpain activity. Accordingly, EGb761-mediated suppression of lipid accumulation in foam cells was also abrogated by HO-1 siRNA. Moreover, the lesion size of atherosclerosis was smaller in EGb761-treated, apolipoprotein E-deficient mice compared with the vehicle-treated mice, and the expression of HO-1, SR-A, and ABCA1 in aortas was modulated similar to that observed in macrophages. CONCLUSION These findings suggest that EGb761 confers a protection from the formation of foam cells by a novel HO-1-dependent regulation of cholesterol homeostasis in macrophages.

Ginkgo biloba extract confers protection from cigarette smoke extract-induced apoptosis in human lung endothelial cells: Role of heme oxygenase-1.

Cigarette smoking is the major cause of chronic obstructive pulmonary disease, which is associated with increased oxidative stress and numbers of apoptotic endothelial cells in the lungs. Ginkgo biloba extract (EGb) is a therapeutic agent for disorders such as vascular insufficiency and Alzheimers disease. Although EGb is known to possess antioxidant functions, its ability to alleviate cigarette smoke-induced pathophysiological consequences has not been elucidated. We investigated the cytoprotective effects and therapeutic mechanisms of EGb against oxidative stress and apoptosis induced by cigarette smoke extract (CSE) in human pulmonary artery endothelial cells (HPAECs). Challenge with CSE (160 microg/ml) caused a reduction in cell viability, an increase in intracellular reactive oxygen species and an acceleration of caspase-dependent apoptosis in HPAECs, all of which were alleviated by pretreatment with EGb (100 microg/ml). N-acetylcysteine (an antioxidant) also reduced both the CSE-induced oxidative stress and apoptosis, indicating that the former response triggered the latter. Additionally, EGb produced activation of ERK, JNK and p38 [three major mitogen-activated protein kinases (MAPKs)], an increase in the nuclear level of nuclear factor erythroid-2-related factor 2 (Nrf2) and upregulation of heme oxygenase-1 (HO-1, a stress-responsive protein with antioxidant function). Pretreatment with inhibitors of MAPKs abolished both EGb-induced Nrf2 nuclear translocation and HO-1 upregulation. Small interfering RNAs targeting HO-1 prevented EGb-induced HO-1 upregulation and also abolished the antioxidant, anti-apoptotic and cytoprotective effects of EGb in HPAECs insulted with CSE. We conclude that EGb confers protection from oxidative stress-related apoptosis induced by CSE in HPAECs and its therapeutic effects depend on transcriptional upregulation of HO-1 by EGb via the MAPKs/Nrf2 pathway.

Valsartan regulates the interaction of angiotensin II type 1 receptor and endothelial nitric oxide synthase via Src/PI3K/Akt signalling

AIMS Valsartan, a selective angiotensin II type 1 receptor (AT1R) blocker, has beneficial effects in the cardiovascular system in part by its increase of nitric oxide (NO) bioavailability, yet the mechanisms are unclear. We investigated the molecular mechanisms underlying this effect in endothelial cells (ECs). METHODS AND RESULTS NO production was examined by Griess reagent assay, DAF-2 DA fluorescence staining and cGMP ELISA kits. Protein interaction was determined by western blotting and immunoprecipitation. Treating bovine or human aortic ECs with valsartan increased NO production, as evidenced by elevated level of stable NO metabolites and intracellular cGMP. Valsartan increased the phosphorylation but not the protein level of endothelial NO synthase (eNOS). Inhibition of phosphoinositide-3 kinase (PI3K)/Akt and Src pathways by specific inhibitors suppressed valsartan-induced NO release. In addition, valsartan increased the tyrosine residue phosphorylation of AT1R, which was attenuated by inhibition of Src but not PI3K activities. Valsartan also suppressed the interaction of eNOS and AT1R, which was blocked by Src or PI3K inhibition. CONCLUSION Valsartan-induced NO production in ECs is mediated through Src/PI3K/Akt-dependent phosphorylation of eNOS. Valsartan-induced AT1R phosphorylation depends on Src but not PI3K, whereas valsartan-induced suppression of AT1R-eNOS interaction depends on Src/PI3K/Akt signalling. These results indicate a novel vasoprotective mechanism of valsartan in upregulating NO production in ECs.

Novel role of AMP-activated protein kinase signaling in cigarette smoke induction of IL-8 in human lung epithelial cells and lung inflammation in mice

Cigarette smoke (CS) increases chemokine production in lung epithelial cells (LECs), but the pathways involved are not completely understood. AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, may modulate inflammation. Here, we show that cigarette smoke extract sequentially activated NADPH oxidase; increased intracellular reactive oxygen species (ROS) level; activated AMPK, NF-κB, and STAT3; and induced interleukin 8 (IL-8) in human LECs. Inhibition of NADPH oxidase activation by apocynin or siRNA targeting p47(phox) (a subunit of NADPH oxidase) attenuated the increased intracellular ROS level, AMPK activation, and IL-8 induction. Removal of intracellular ROS by N-acetylcysteine reduced the AMPK activation and IL-8 induction. Prevention of AMPK activation by Compound C or AMPK siRNA lessened the activation of both NF-κB and STAT3 and the induction of IL-8. Abrogation of the activation of NF-κB and STAT3 by BAY11-7085 and AG490, respectively, attenuated the IL-8 induction. We additionally show that chronic CS exposure in mice promoted AMPK phosphorylation and expression of MIP-2α (an IL-8 homolog) in LECs and lungs, as well as lung inflammation, all of which were reduced by Compound C treatment. Thus, a novel NADPH oxidase-dependent, ROS-sensitive AMPK signaling is important for CS-induced IL-8 production in LECs and possibly lung inflammation.

Wood smoke extract promotes both apoptosis and proliferation in rat alveolar epithelial type II cells: the role of oxidative stress and heme oxygenase-1.

Objective:Inhalation of toxic smoke causes oxidant lung injury. Alveolar epithelial type II cells are important in the re-epithelialization of alveolar walls after lung injury. We investigated the responses of alveolar epithelial type II cells to insult by wood smoke extract, and we identified the role of reactive oxygen species and heme oxygenase-1 (an oxidative stress protein) in these responses. Design:A randomized, controlled study. Setting:A research laboratory. Subjects:Cultured rat L2 and primary alveolar epithelial type II cells. Interventions and Main Results:Exposure of L2 alveolar epithelial type II cells to smoke extract (60 &mgr;g/mL) caused increases in reactive oxygen species, mitogen-activated protein kinases phosphylation, heme oxygenase-1 expression, apoptosis, proliferation and cell population, all of which were largely reduced by N-acetylcysteine (an antioxidant). Additionally, the smoke extract-induced heme oxygenase-1 induction was significantly attenuated by mitogen-activated protein kinases inhibitors, by small interfering RNA targeting mitogen-activated protein kinases or by N-acetylcysteine. Furthermore, knockdown of heme oxygenase-1 by small interfering RNA prevented heme oxygenase-1 induction whereas increasing smoke extract-induced apoptosis and suppressing smoke extract-induced proliferation. Conversely, cobalt protoporphyrin IX (a heme oxygenase-1 inducer) amplified heme oxygenase-1 induction while suppressing smoke extract-induced apoptosis and augmenting smoke extract-induced proliferation. Consequently, the smoke extract-induced increase in cell population was changed into a decrease by heme oxygenase-1 small interfering RNA, but was further elevated by cobalt protoporphyrin IX. Smoke extract also caused increases in heme oxygenase-1 expression, apoptosis, proliferation and cell population in primary alveolar epithelial type II cells, and heme oxygenase-1 small interfering RNA similarly augmented smoke extract-induced apoptosis and suppressed smoke extract-induced proliferation in these primary cells. Conclusions:Smoke extract increases intracellular reactive oxygen species, which up-regulates heme oxygenase-1 via the mitogen-activated protein kinase pathways and also promotes both apoptosis and proliferation in rat alveolar epithelial type II cells. Additionally, smoke extract-induced heme oxygenase-1 induction counteracts smoke extract-induced apoptosis, but mediates smoke extract-induced proliferation, resulting in a net increase in cell population. Thus, in response to oxidant smoke insult, alveolar epithelial type II cells have evolved an adaptive mechanism involving heme oxygenase-1 that increases their cell population, presumably to help them perform their function of re-epithelialization following lung injury.

Glucosamine attenuates cigarette smoke-induced lung inflammation by inhibiting ROS-sensitive inflammatory signaling

Cigarette smoking causes persistent lung inflammation that is mainly regulated by redox-sensitive pathways. We have reported that cigarette smoke (CS) activates a NADPH oxidase-dependent reactive oxygen species (ROS)-sensitive AMP-activated protein kinase (AMPK) signaling pathway leading to induction of lung inflammation. Glucosamine, a dietary supplement used to treat osteoarthritis, has antioxidant and anti-inflammatory properties. However, whether glucosamine has similar beneficial effects against CS-induced lung inflammation remains unclear. Using a murine model we show that chronic CS exposure for 4 weeks increased lung levels of 4-hydroxynonenal (an oxidative stress biomarker), phospho-AMPK, and macrophage inflammatory protein 2 and induced lung inflammation; all of these CS-induced events were suppressed by chronic treatment with glucosamine. Using human bronchial epithelial cells, we demonstrate that cigarette smoke extract (CSE) sequentially activated NADPH oxidase; increased intracellular levels of ROS; activated AMPK, mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription proteins 3 (STAT3); and induced interleukin-8 (IL-8). Additionally, using a ROS scavenger, a siRNA that targets AMPK, and various pharmacological inhibitors, we identified the signaling cascade that leads to induction of IL-8 by CSE. All these CSE-induced events were inhibited by glucosamine pretreatment. Our findings suggest a novel role for glucosamine in alleviating the oxidative stress and lung inflammation induced by chronic CS exposure in vivo and in suppressing the CSE-induced IL-8 in vitro by inhibiting both the ROS-sensitive NADPH oxidase/AMPK/MAPK signaling pathway and the downstream transcriptional factors NF-κB and STAT3.

α-Lipoic acid ameliorates foam cell formation via liver X receptor α-dependent upregulation of ATP-binding cassette transporters A1 and G1

α-Lipoic acid (α-LA), a key cofactor in cellular energy metabolism, has protective activities in atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined whether α-LA affects foam cell formation and its underlying molecular mechanisms in murine macrophages. Treatment with α-LA markedly attenuated oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, which was due to increased cholesterol efflux. Additionally, α-LA treatment dose-dependently increased protein levels of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 but had no effect on the protein expression of SR-A, CD36, or SR-BI involved in cholesterol homeostasis. Furthermore, α-LA increased the mRNA expression of ABCA1 and ABCG1. The upregulation of ABCA1 and ABCG1 by α-LA depended on liver X receptor α (LXRα), as evidenced by an increase in the nuclear levels of LXRα and LXRE-mediated luciferase activity and its prevention of the expression of ABCA1 and ABCG1 after inhibition of LXRα activity by the pharmacological inhibitor geranylgeranyl pyrophosphate (GGPP) or knockdown of LXRα expression with small interfering RNA (siRNA). Consistently, α-LA-mediated suppression of oxLDL-induced lipid accumulation was abolished by GGPP or LXRα siRNA treatment. In conclusion, LXRα-dependent upregulation of ABCA1 and ABCG1 may mediate the beneficial effect of α-LA on foam cell formation.

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009.

Statins are associated with a reduced risk of cholangiocarcinoma: a population‐based case–control study

AIMS Cholangiocarcinoma (CCA) is the second most common primary liver cancer in the world. Due to the lack of effective treatments, the survival rate of CCA is low and it is usually considered difficult to diagnose early. To date, no effective strategies for the prevention of CCA have been developed. Statins are cholesterol-lowering agents which possess pleiotropic properties and the use of statins may reduce cancer risk. The aim of the study was to investigate the effect of statin use on the risk of CCA. METHODS We used nationwide insurance data to perform a case-control study including 3174 CCA patients diagnosed in 2002-2011 and 3174 propensity score matched controls. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated to assess the association between CCA risk and statin use by type of statin and dose. RESULTS Patients with CCA were slightly younger than controls with mean ages of 67.4 (SD 12.3) and 68.5 (SD 13.2) years (P = 0.001), respectively, and had less users of statins (22.7 vs. 26.5%, P < 0.001). The overall adjusted OR of statin use associated CCA was 0.80 (95% CI 0.71, 0.90) and lowered for those with longer medications. The OR ranged from 0.65 to 0.77. Stronger dose-response association was seen when using lovastatin. CONCLUSIONS Statin use is associated with reduced risk of CCA and there is a dose-response relationship between the use of statins and risk of CCA.