Song-Kun Shyue

15d-Prostaglandin J2 Protects Brain From Ischemia-Reperfusion Injury

Objective—Brain expresses abundant lipocalin-type prostaglandin (PG) D2 (PGD2) synthase but the role of PGD2 and its metabolite, 15-deoxy-&Dgr;12,14 PGJ2 (15d-PGJ2) in brain protection is unclear. The aim of this study is to assess the effect of 15d-PGJ2 on neuroprotection. Methods and Results—Adenoviral transfer of cyclooxygenase-1 (Adv-COX-1) was used to amplify the production of 15d-PGJ2 in ischemic cortex in a rat focal infarction model. Cortical 15d-PGJ2 in Adv-COX-1–treated rats was increased by 3-fold over control, which was correlated with reduced infarct volume and activated caspase 3, and increased peroxisome proliferator activated receptor-&ggr; (PPAR&ggr;) and heme oxygenase-1 (HO-1). Intraventricular infusion of 15d-PGJ2 resulted in reduction of infarct volume, which was abrogated by a PPAR&ggr; inhibitor. Rosiglitazone infusion had a similar effect. 15d-PGJ2 and rosiglitazone at low concentrations suppressed H2O2-induced rat or human neuronal apoptosis and necrosis and induced PPAR&ggr; and HO-1 expression. The anti-apoptotic effect was abrogated by PPAR&ggr; inhibition. Conclusion—15d-PGJ2 suppressed ischemic brain infarction and neuronal apoptosis and necrosis in a PPAR&ggr; dependent manner. 15d-PGJ2 may play a role in controlling acute brain damage induced by ischemia-reperfusion.

Superoxide Dismutase Inhibits the Expression of Vascular Cell Adhesion Molecule-1 and Intracellular Cell Adhesion Molecule-1 Induced by Tumor Necrosis Factor-α in Human Endothelial Cells Through the JNK/p38 Pathways

Objective— Expression of adhesion molecules on endothelial cells and subsequent leukocyte recruitment are critical early events in the development of atherosclerosis. We tried to study possible effects of Cu/Zn superoxide dismutase (SOD) on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders. Methods and Results— Human aortic endothelial cells (HAECs) were transfected with adenovirus carrying the human SOD gene (AdSOD) to investigate whether SOD expression in HAECs attenuated tumor necrosis factor (TNF)-&agr;–induced reactive oxygen species production and adhesion molecule expression and to define the mechanisms involved. SOD expression significantly suppressed TNF-&agr;–induced expression of vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1 and reduced the binding of the human neutrophils to TNF-&agr;–stimulated HAECs. SOD expression suppressed c-JUN N-terminal kinase and p38 phosphorylation. It also attenuated intracellular superoxide anion production and NADPH oxidase activity in TNF-&agr;–treated HAECs. Conclusions— These results provide evidence that SOD expression in endothelial cells attenuates TNF-&agr;–induced superoxide anion production and adhesion molecule expression, and that this protective effect is mediated by decreased JNK and p38 phosphorylation and activator protein-1 and nuclear factor &kgr;B inactivation. These results suggest that SOD has antiinflammatory properties and may play important roles in the prevention of atherosclerosis and inflammatory response.

Ligand-Activated Peroxisome Proliferator–Activated Receptor-γ Protects Against Ischemic Cerebral Infarction and Neuronal Apoptosis by 14-3-3ε Upregulation

Background— Thiazolidinediones have been reported to protect against ischemia-reperfusion injury. Their protective actions are considered to be peroxisome proliferator–activated receptor-γ (PPAR-γ)–dependent; however, it is unclear how PPAR-γ activation confers resistance to ischemia-reperfusion injury. Methods and Results— We evaluated the effects of rosiglitazone or PPAR-γ overexpression on cerebral infarction in a rat model and investigated the antiapoptotic actions in the N2-A neuroblastoma cell model. Rosiglitazone or PPAR-γ overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR-γ small interfering RNA. In mice with knock-in of a PPAR-γ dominant-negative mutant, infarct volume was enhanced. Proteomic analysis revealed that brain 14-3-3&egr; was highly upregulated in rats treated with rosiglitazone. Upregulation of 14-3-3&egr; was abrogated by PPAR-γ small interfering RNA or antagonist. Promoter analysis and chromatin immunoprecipitation revealed that rosiglitazone induced PPAR-γ binding to specific regulatory elements on the 14-3-3&egr; promoter and thereby increased 14-3-3&egr; transcription. 14-3-3&egr; Small interfering RNA abrogated the antiapoptotic actions of rosiglitazone or PPAR-γ overexpression, whereas 14-3-3&egr; recombinant proteins rescued brain tissues and N2-A cells from ischemia-induced damage and apoptosis. Elevated 14-3-3&egr; enhanced binding of phosphorylated Bad and protected mitochondrial membrane potential. Conclusions— Ligand-activated PPAR-γ confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3&egr; transcription. 14-3-3&egr; Upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis.

β Common receptor integrates the erythropoietin signaling in activation of endothelial nitric oxide synthase.

Erythropoietin (EPO), the key hormone for erythropoiesis, also increases nitric oxide (NO) bioavailability in endothelial cells (ECs), yet the definitive mechanisms are not fully understood. Increasing evidence has demonstrated that β common receptor (βCR) plays a crucial role in EPO‐mediated non‐hematopoietic effects. We investigated the role of βCR in EPO‐induced endothelial NO synthase (eNOS) activation in bovine aortic ECs (BAECs) and the molecular mechanisms involved. Results of confocal microscopy and immunoprecipitation analyses revealed that βCR was colocalized and interacted with EPO receptor (EPOR) in ECs. Inhibition of βCR or EPOR by neutralizing antibodies or small interfering RNA abolished the EPO‐induced NO production. Additionally, blockage of βCR abrogated the EPO‐induced increase in the phosphorylation of eNOS, Akt, Src, or Janus kinase 2 (JAK2). Immunoprecipitation analysis revealed that treatment with EPO increased the interaction between βCR and eNOS, which was suppressed by inhibition of Src, JAK2, or Akt signaling with specific pharmacological inhibitors. Furthermore, EPO‐induced EC proliferation, migration, and tube formation were blocked by pretreatment with βCR antibody and Src, JAK2, or PI3K/Akt inhibitors. Moreover, in vivo experiments showed that EPO increased the level of phosphorylated eNOS, Src, JAK2, and Akt, as well as βCR–eNOS association in aortas and promoted the angiogenesis in Matrigel plug, which was diminished by βCR or EPOR neutralizing antibodies. Our findings suggest that βCR may play an integrative role in the EPO signaling‐mediated activation of eNOS in ECs. J. Cell. Physiol. 226: 3330–3339, 2011.

Inhibition of cadmium‐induced oxidative injury in rat primary astrocytes by the addition of antioxidants and the reduction of intracellular calcium

Exposure of the brain to cadmium ions (Cd2+) is believed to lead to neurological disorders of the central nervous system (CNS). In this study, we tested the hypothesis that astrocytes, the major CNS‐supporting cells, are resistant to Cd2+‐induced injury compared with cortical neurons and microglia (CNS macrophages). However, treatment with CdCl2 for 24 h at concentrations higher than 20 µM substantially induced astrocytic cytotoxicity, which also resulted from long‐term exposure to 5 µM of CdCl2. Intracellular calcium levels were found to rapidly increase after the addition of CdCl2 into astrocytes, which led to a rise in reactive oxygen species (ROS) and to mitochondrial impairment. In accordance, preexposure to the extracellular calcium chelator EGTA effectively reduced ROS production and increased survival of Cd2+‐treated astrocytes. Adenovirus‐mediated transfer of superoxide dismutase (SOD) or glutathione peroxidase (GPx) genes increased survival of Cd2+‐exposed astrocytes. In addition, increased ROS generation and astrocytic cell death due to Cd2+ exposure was inhibited when astrocytes were treated with the polyphenolic compound ellagic acid (EA). Taken together, Cd2+‐induced astrocytic cell death resulted from disrupted calcium homeostasis and an increase in ROS. Moreover, our findings demonstrate that enhancement of the activity of intracellular antioxidant enzymes and supplementation with a phenolic compound, a natural antioxidant, improves survival of Cd2+‐primed astrocytes. This information provides a useful approach for treating Cd2+‐induced CNS neurological disorders. J. Cell. Biochem. 103: 825–834, 2008.

Molecular mechanism of curcumin on the suppression of cholesterol accumulation in macrophage foam cells and atherosclerosis

SCOPE Curcumin, a potent antioxidant extracted from Curcuma longa, confers protection against atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined the effect of curcumin on lipid accumulation and the underlying molecular mechanisms in macrophages and apolipoprotein E-deficient (apoE⁻/⁻) mice. METHODS AND RESULTS Treatment with curcumin markedly ameliorated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages, which was due to decreased oxLDL uptake and increased cholesterol efflux. In addition, curcumin decreased the protein expression of scavenger receptor class A (SR-A) but increased that of ATP-binding cassette transporter (ABC) A1 and had no effect on the protein expression of CD36, class B receptor type I (SR-BI), or ATP-binding cassette transporter G1 (ABCG1). The downregulation of SR-A by curcumin was via ubiquitin-proteasome-calpain-mediated proteolysis. Furthermore, the curcumin-induced upregulation of ABCA1 was mainly through calmodulin-liver X receptor α (LXRα)-dependent transcriptional regulation. Curcumin administration modulated the expression of SR-A, ABCA1, ABCG1, and SR-BI in aortas and retarded atherosclerosis in apoE⁻/⁻ mice. CONCLUSION Our findings suggest that inhibition of SR-A-mediated oxLDL uptake and promotion of ABCA1-dependent cholesterol efflux are two crucial events in suppression of cholesterol accumulation by curcumin in the transformation of macrophage foam cells.

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.

Essential role of transient receptor potential vanilloid type 1 in evodiamine-mediated protection against atherosclerosis

We investigated whether transient receptor potential vanilloid type 1 (TRPV1) was involved in the therapeutic effect of evodiamine, a main bioactive component in the fruit of Evodiae rutaecarpa, on the development of atherosclerosis in apolipoprotein E‐deficient (ApoE−/−) mice and ApoE−/−TRPV1−/− mice.

Stabilization of Hypoxia-inducible Factor-1α by Prostacyclin under Prolonged Hypoxia via Reducing Reactive Oxygen Species Level in Endothelial Cells

Hypoxia-inducible factor-1 (HIF-1) takes part in the transcriptional activation of hypoxia-responsive genes. HIF-1α, a subunit of HIF-1, is rapidly degraded under normoxic conditions by the ubiquitin-proteosome system. Hypoxia up-regulates HIF-1α by inhibiting its degradation, thereby allowing it to accumulate to high levels with 3–6 h of hypoxia treatment and decreasing thereafter. In vascular tissues, prostacyclin (prostaglandin I2 (PGI2)) is a potent vasodilator and inhibitor of platelet aggregation and is known as a vasoprotective molecule. However, the role of PGI2 in HIF-1 activation has not been studied. In the present study, we investigated the effect of PGI2 on HIF-1 regulation in human umbilical vein endothelial cells under prolonged hypoxia (12 h). Augmentation of PGI2 via adenovirus-mediated gene transfer of both cyclooxygenase-1 and PGI2 synthase activated HIF-1 by stabilizing HIF-1α in cells under prolonged hypoxia or the hypoxia-normoxia transition but not under normoxia. Exogenous H2O2 abolished PGI2- and catalase-induced HIF-1α up-regulation, which suggests that degradation of HIF-1α under prolonged hypoxia is through a reactive oxygen species-dependent pathway. Moreover, PGI2 attenuated NADPH oxidase activity by suppressing Rac1 and p47phox expression under hypoxia. These data demonstrate a novel function of PGI2 in down-regulating reactive oxygen species production by attenuating NADPH oxidase activity, which stabilizes HIF-1α in human umbilical vein endothelial cells exposed to prolonged hypoxia.

Induction of prostacyclin/PGI2 synthase expression after cerebral ischemia-reperfusion.

Prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet aggregation and leukocyte activation, is crucial in vascular diseases such as stroke. Prostacyclin synthase (PGIS) is the key enzyme for PGI2 synthesis. Although expression of PGIS was noted in the brain, its role in ischemic insult remains unclear. Here we reported the temporal and spatial expression of PGIS mRNA and protein after 60-min transient ischemia. Northern blot and in situ hybridization revealed a delayed increase of PGIS mRNA in the ischemic cortex at 24- to 72-h after ischemia; PGIS was detected mainly in the ipsilateral penumbra area, pyriform cortex, hippocampus, and leptomeninges. Western blot and immunohistochemical analysis revealed that PGIS proteins were expressed temporally and spatially similar to PGIS mRNA. PGIS was heavily colocalized with PECAM-1 to endothelial cells at the leptomeninges, large and small vessels, and localized to neuronal cells, largely at the penumbra area. A substantial amount of PGIS was also detected in the macrophage and glial cells. To evaluate its role against ischemic infarct, we overexpressed PGIS by adenoviral gene transfer. When infused 72 h before ischemia (–72 h), Adv-PGIS reduced infarct volume by ~50%. However, it had no effect on infarct volume when infused immediately after ischemia (0 h). Eicosanoid analysis revealed selective elevation of PGI2 at −72 h while PGI2 and TXB2 were both elevated at 0 h, altering the PGI2/thromboxane A2 (TXA2) ratio from 10 to 4. These findings indicate that PGIS protects the brain by enhancing PGI2 synthesis and creating a favorable PGI2/TXA2 ratio.