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Find video protocols related to scientific articles indexed in Pubmed.
CD137-inducing factors from T cells and macrophages accelerate the destabilization of atherosclerotic plaques in hyperlipidemic mice.
FASEB J.
PUBLISHED: 07-24-2014
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CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, has been reported to be expressed in atherosclerotic plaques, and to promote lesion formation. However, the role of CD137 in mediating atherosclerotic plaque stability and the possible underlying molecular and cellular mechanisms are poorly understood. Here, apolipoprotein E-deficient (ApoE(-/-)) and CD137-deficient ApoE(-/-) (ApoE(-/-)CD137(-/-)) mice fed a chow diet for 66 wk were used. CD137 induces plaque instability, which is characterized by increased plaque necrosis, decreased collagen content, decreased vascular smooth muscle cell (VSMC) content, and increased macrophage infiltration. CD137 also increases the infiltration of effector T (Teff) cells into plaque lesion sites, resulting in increased interferon-? (IFN-?) expression. Interestingly, Teff-cell-derived IFN-? inhibits collagen synthesis in atherosclerotic plaques. Furthermore, CD137 activation increases the apoptosis of VSMCs, possibly by decreasing the antiapoptotic regulator, Bcl-2, and subsequently up-regulating cleaved caspase-3. In macrophages, activation of CD137 signaling boosted the oxidized low density lipoprotein-induced expression of matrix metalloproteinase 9 via the p38 mitogen-activated protein kinase and extracellular signal-regulated kinase1/2 signaling pathways. In summary, activation of CD137 signaling decreases the stability of advanced atherosclerotic plaques via its combined effects on Teff cells, VSMCs, and macrophages.-Jung, I.-H., Choi, J.-H., Jin, J., Jeong, S.-J., Jeon, S., Lim, C., Lee, M.-R., Yoo, J.-Y., Sonn, S.-K., Kim, Y. H., Choi, B. K., Kwon, B. S., Seoh, J.-Y., Lee, C. W., Kim, D.-Y., Oh, G. T. CD137-inducing factors from T cells and macrophages accelerate the destabilization of atherosclerotic plaques in hyperlipidemic mice.
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Inhibition of Ninjurin 1 restores erectile function through dual angiogenic and neurotrophic effects in the diabetic mouse.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-16-2014
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Penile erection is a neurovascular phenomenon, and erectile dysfunction (ED) is caused mainly by vascular risk factors or diseases, neurologic abnormalities, and hormonal disturbances. Men with diabetic ED often have severe endothelial dysfunction and peripheral nerve damage, which result in poor response to oral phosphodiesterase-5 inhibitors. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. Here, we demonstrate in streptozotocin-induced diabetic mice that inhibition of the Ninj1 pathway by administering Ninj1-neutralizing antibody (Ninj1-Ab) or by using Ninj1-knockout mice successfully restored erectile function through enhanced penile angiogenesis and neural regeneration. Angiopoietin-1 (Ang1) expression was down-regulated and angiopoietin-2 expression was up-regulated in the diabetic penis compared with that in controls, and these changes were reversed by treatment with Ninj1-Ab. Ninj1 blockade-mediated penile angiogenesis and neural regeneration as well as recovery of erectile function were abolished by inhibition of Ang1-Tie2 (tyrosine kinase with Ig and epidermal growth factor homology domain-2) signaling with soluble Tie2 antibody or Ang1 siRNA. The present results suggest that inhibition of the Ninj1 pathway will be a novel therapeutic strategy for treating ED.
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Ninjurin1 enhances the basal motility and transendothelial migration of immune cells by inducing protrusive membrane dynamics.
J. Biol. Chem.
PUBLISHED: 06-10-2014
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Ninjurin1 is involved in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, by mediating leukocyte extravasation, a process that depends on homotypic binding. However, the precise regulatory mechanisms of Ninjurin1 during inflammation are largely undefined. We therefore examined the pro-migratory function of Ninjurin1 and its regulatory mechanisms in macrophages. Interestingly, Ninjurin1-deficient bone marrow-derived macrophages exhibited reduced membrane protrusion formation and dynamics, resulting in the impairment of cell motility. Furthermore, exogenous Ninjurin1 was distributed at the membrane of filopodial structures in Raw264.7 macrophage cells. In Raw264.7 cells, RNA interference of Ninjurin1 reduced the number of filopodial projections, whereas overexpression of Ninjurin1 facilitated their formation and thus promoted cell motility. Ninjurin1-induced filopodial protrusion formation required the activation of Rac1. In Raw264.7 cells penetrating an MBEC4 endothelial cell monolayer, Ninjurin1 was localized to the membrane of protrusions and promoted their formation, suggesting that Ninjurin1-induced protrusive activity contributed to transendothelial migration. Taking these data together, we conclude that Ninjurin1 enhances macrophage motility and consequent extravasation of immune cells through the regulation of protrusive membrane dynamics. We expect these findings to provide insight into the understanding of immune responses mediated by Ninjurin1.
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NAA10 controls osteoblast differentiation and bone formation as a feedback regulator of Runx2.
Nat Commun
PUBLISHED: 05-22-2014
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Runt-related transcription factor 2 (Runx2) transactivates many genes required for osteoblast differentiation. The role of N-?-acetyltransferase 10 (NAA10, arrest-defective-1), originally identified in yeast, remains poorly understood in mammals. Here we report a new NAA10 function in Runx2-mediated osteogenesis. Runx2 stabilizes NAA10 in osteoblasts during BMP-2-induced differentiation, and NAA10 in turn controls this differentiation by inhibiting Runx2. NAA10 delays bone healing in a rat calvarial defect model and bone development in neonatal mice. Mechanistically, NAA10 acetylates Runx2 at Lys225, and this acetylation inhibits Runx2-driven transcription by interfering with CBF? binding to Runx2. Our study suggests that NAA10 acts as a guard ensuring balanced osteogenesis by fine-tuning Runx2 signalling in a feedback manner. NAA10 inhibition could be considered a potential strategy for facilitating bone formation.
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Hypoxia-induced endothelial progenitor cell function is blunted in angiotensinogen knockout mice.
Mol. Cells
PUBLISHED: 05-09-2014
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Angiotensinogen (AGT), the precursor of angiotensin I, is known to be involved in tumor angiogenesis and associated with the pathogenesis of coronary atherosclerosis. This study was undertaken to determine the role played by AGT in endothelial progenitor cells (EPCs) in tumor progression and metastasis. It was found that the number of EPC colonies formed by AGT heterozygous knockout (AGT(+/-)) cells was less than that formed by wild-type (WT) cells, and that the migration and tube formation abilities of AGT(+/-) EPCs were significantly lower than those of WT EPCs. In addition, the gene expressions of vascular endothelial growth factor (VEGF), Flk1, angiopoietin (Ang)-1, Ang-2, Tie-2, stromal derived factor (SDF)-1, C-X-C chemokine receptor type 4 (CXCR4), and of endothelial nitric oxide synthase (eNOS) were suppressed in AGT(+/-) EPCs. Furthermore, the expressions of hypoxia-inducible factor (HIF)-1? and -2? were downregulated in AGT(+/-) early EPCs under hypoxic conditions, suggesting a blunting of response to hypoxia. Moreover, the activation of Akt/eNOS signaling pathways induced by VEGF, epithelial growth factor (EGF), or SDF-1? were suppressed in AGT(+/-) EPCs. In AGT(+/-) mice, the incorporation of EPCs into the tumor vasculature was significantly reduced, and lung tumor growth and melanoma metastasis were attenuated. In conclusion, AGT is required for hypoxia-induced vasculogenesis.
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The adipokine Retnla modulates cholesterol homeostasis in hyperlipidemic mice.
Nat Commun
PUBLISHED: 04-26-2014
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Hyperlipidemia is a well-recognized risk factor for atherosclerosis and can be regulated by adipokines. Expression of the adipokine resistin-like molecule alpha (Retnla) is regulated by food intake; whether Retnla has a role in the pathogenesis of hyperlipidemia and atherosclerosis is unknown. Here we report that Retnla has a cholesterol-lowering effect and protects against atherosclerosis in low-density lipoprotein receptor-deficient mice. On a high-fat diet, Retnla deficiency promotes hypercholesterolaemia and atherosclerosis, whereas Retnla overexpression reverses these effects and improves the serum lipoprotein profile, with decreased cholesterol in the very low-density lipoprotein fraction concomitant with reduced serum apolipoprotein B levels. We show that Retnla upregulates cholesterol-7-?-hydroxylase, a key hepatic enzyme in the cholesterol catabolic pathway, through induction of its transcriptional activator liver receptor homologue-1, leading to increased excretion of cholesterol in the form of bile acids. These findings define Retnla as a novel therapeutic target for treating hypercholesterolaemia and atherosclerosis.
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Two distinct domains of Flo8 activator mediates its role in transcriptional activation and the physical interaction with Mss11.
Biochem. Biophys. Res. Commun.
PUBLISHED: 04-21-2014
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Flo8 is a transcriptional activator essential for the inducible expression of a set of target genes such as STA1, FLO11, and FLO1 encoding an extracellular glucoamylase and two cell surface proteins, respectively. However, the molecular mechanism of Flo8-mediated transcriptional activation remains largely elusive. By generating serial deletion constructs, we revealed here that a novel transcriptional activation domain on its extreme C-terminal region plays a crucial role in activating transcription. On the other hand, the N-terminal LisH motif of Flo8 appears to be required for its physical interaction with another transcriptional activator, Mss11, for their cooperative transcriptional regulation of the shared targets. Additionally, GST pull-down experiments uncovered that Flo8 and Mss11 can directly form either a heterodimer or a homodimer capable of binding to DNA, and we also showed that this formed complex of two activators interacts functionally and physically with the Swi/Snf complex. Collectively, our findings provide valuable clues for understanding the molecular mechanism of Flo8-mediated transcriptional control of multiple targets.
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Developmental endothelial locus-1 inhibits MIF production through suppression of NF-?B in macrophages.
Int. J. Mol. Med.
PUBLISHED: 01-22-2014
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Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that regulates leukocyte recruitment, thereby playing a pivotal role in the regulation of innate and adaptive immunity and tumor progression. Elevated levels of MIF are associated with numerous inflammatory disorders and cancers. To determine whether developmental endothelial locus-1 (Del-1) regulated MIF, RAW264.7 macrophages were treated with Del-1 and assessed using ELISA. The results showed that MIF was downregulated in macrophages by Del-1, an endogenous anti-inflammatory protein that was previously shown to limit leukocyte adhesion and migration. Treatment of RAW264.7 macrophages with Del-1 inhibited constitutive and lipopolysaccharide (LPS)-induced MIF secretion. Recombinant Del-1 protein attenuated the phosphorylation of I?B? induced by a relatively low concentration of LPS in THP-1 monocytes, but did not inhibit I?B? phosphorylation in response to a relatively high concentration of LPS. Concomitantly, translocation of NF-?B to the nucleus was inhibited by Del-1 in LPS-activated macrophages. In addition, conditioned medium harvested from cells transfected with a Del-1 expression plasmid suppressed NF-?B activation in response to relatively low concentrations of TNF-?, albeit not the activation that was induced by a relatively high concentration of TNF-?. On the other hand, although Del-1 enhanced the macrophage expression of p53, a known negative regulator of MIF production, MIF production was not significantly affected by the level of p53 in mouse bone marrow-derived macrophages. These findings suggested that Del-1 controls NF-?B-activated MIF production in macrophages, and the potential application of Del-1 to therapeutic modalities for chronic inflammation-associated cancers.
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Tripartite motif-containing protein 30 modulates TCR-activated proliferation and effector functions in CD4+ T cells.
PLoS ONE
PUBLISHED: 01-01-2014
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To avoid excessive activation, immune signals are tightly controlled by diverse inhibitory proteins. TRIM30, a tripartite motif (TRIM)-containing protein is one of such inhibitors known to function in macrophages. To define the roles of TRIM30, we generated Trim30 knockout (Trim30-/-) mice. Trim30 deletion caused no major developmental defects in any organs, nor showed any discernable defect in the activation of macrophages. But, Trim30-/- mice showed increased CD4/CD8 ratio when aged and Trim30-/- CD4+ T cells exhibited an abnormal response upon TCR activation, in particular in the absence of a costimulatory signal. Adoptive transfer of wild-type and Trim30-/- CD4+ T cells together into lymphopenic hosts confirmed higher proliferation of the Trim30-/- CD4+ T cells in vivo. Despite the enhanced proliferation, Trim30-/- T cells showed decreased levels of NF-?B activation and IL-2 production compared to wild-type cells. These results indicate a distinct requirement for TRIM30 in modulation of NF-?B activation and cell proliferation induced by TCR stimulation.
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Ninjurin1 deficiency attenuates susceptibility of experimental autoimmune encephalomyelitis in mice.
J. Biol. Chem.
PUBLISHED: 12-17-2013
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Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple sclerosis (MS). However, in vivo gene-deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knockout (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis (EIU). Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in EIU mice. Moreover, the administration of a custom-made antibody (Ab26-37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone-marrow derived macrophages (BMDMs) and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO BMDMs and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1 overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as MS.
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p53 regulates the transcription of the anti-inflammatory molecule developmental endothelial locus-1 (Del-1).
Oncotarget
PUBLISHED: 11-07-2013
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Developmental endothelial locus-1 (Del-1) is an endothelium-derived anti-inflammatory molecule that is downregulated by inflammatory stimuli. Little is known about the molecular mechanisms by which Del-1 transcription is regulated. In the present study, a DNA sequence upstream of the Del-1 gene was analyzed and putative p53 response elements (p53REs) were identified. An approximately 2 kb fragment upstream of the translation start site displayed the highest Del-1 transcriptional activity, and the transcriptional activity of this fragment was enhanced by overexpression of p53. Chemical activation of endogenous p53 elevated the levels of Del-1 mRNA. Site-directed mutagenesis of CATG in the consensus sequences of the 2 kb fragment to TATA significantly reduced the transcription of Del-1. Chromatin immunoprecipitation revealed recruitment of p53 to the p53REs of the Del-1 promoter, resulting in increased Del-1 transcription. Finally, primary endothelial cells isolated from mice with reduced levels of p53 showed a decrease in Del-1 mRNA compared to wild-type endothelial cells. Moreover, Del-1 reciprocally enhanced p53 expression in primary endothelial cells. Thus, these findings suggest that Del-1 is a novel transcriptional target gene of p53.
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Korean red ginseng inhibits arginase and contributes to endotheliumdependent vasorelaxation through endothelial nitric oxide synthase coupling.
J Ginseng Res
PUBLISHED: 05-30-2013
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Korean red ginseng water extract (KG-WE) has known beneficial effects on the cardiovascular system via inducting nitric oxide (NO) production in endothelium. Endothelial arginase inhibits the activity of endothelial nitric oxide synthase (eNOS) by substrate depletion, thereby reducing NO bioavailability and contributing to vascular diseases including hypertension, aging, and atherosclerosis. In the present study, we demonstrate that KG-WE inhibits arginase activity and negatively regulates NO production and reactive oxygen species generation in endothelium. This is associated with increased dimerization of eNOS without affecting the protein expression levels of either arginase or eNOS. In a vascular tension assay, when aortas isolated from wild type mice were incubated with KG-WE, NO-dependent enhanced vasorelaxation was observed. Furthermore, KG-WE administered via by drinking water to atherogenic model mice being fed high cholesterol diet improved impaired vascular function. Taken together, these results suggest that KG-WE may exert vasoprotective effects through augmentation of NO signaling by inhibiting arginase. Therefore, KG-WE may be useful in the treatment of vascular diseases derived from endothelial dysfunction, such as atherosclerosis.
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Identification and characterization of CW108F, a novel ?-carboline compound that promotes cardiomyogenesis of stem cells.
Life Sci.
PUBLISHED: 05-06-2013
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The aim of this study was to identify new compounds that induce cardiomyocyte differentiation of stem cells through cell-based screening and investigate lineage specificity and mechanisms in vitro.
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OASL1 inhibits translation of the type I interferon-regulating transcription factor IRF7.
Nat. Immunol.
PUBLISHED: 02-17-2013
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The production of type I interferon is essential for viral clearance but is kept under tight control to avoid unnecessary tissue damage from hyperinflammatory responses. Here we found that OASL1 inhibited translation of IRF7, the master transcription factor for type I interferon, and thus negatively regulated the robust production of type I interferon during viral infection. OASL1 inhibited the translation of IRF7 mRNA by binding to the 5 untranslated region (UTR) of IRF7 and possibly by inhibiting scanning of the 43S preinitiation complex along the message. Oasl1-/- mice were resistant to viral infection because of the greater abundance of type I interferon, which suggests that OASL1 could be a potential therapeutic target for boosting the production of type I interferon during viral infection.
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Inactivation of max-interacting protein 1 induces renal cilia disassembly through reduction in levels of intraflagellar transport 20 in polycystic kidney.
J. Biol. Chem.
PUBLISHED: 01-13-2013
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Cilia in ciliated cells consist of protruding structures that sense mechanical and chemical signals from the extracellular environment. Cilia are assembled with variety molecules via a process known as intraflagellar transport (IFT). What controls the length of cilia in ciliated cells is critical to understand ciliary disease such as autosomal dominant polycystic kidney disease, which involves abnormally short cilia. But this control mechanism is not well understood. Previously, multiple tubular cysts have been observed in the kidneys of max-interacting protein 1 (Mxi1)-deficient mice aged 6 months or more. Here, we clarified the relationship between Mxi1 inactivation and cilia disassembly. Cilia phenotypes were observed in kidneys of Mxi1-deficient mice using scanning electron microscopy to elucidate the effect of Mxi1 on renal cilia phenotype, and cilia disassembly was observed in Mxi1-deficient kidney. In addition, genes related to cilia were validated in vitro and in vivo using quantitative PCR, and Ift20 was selected as a candidate gene in this study. The length of cilium decreased, and p-ERK level induced by a cilia defect increased in kidneys of Mxi1-deficient mice. Ciliogenesis of Mxi1-deficient mouse embryonic fibroblasts (MEFs) decreased, and this abnormality was restored by Mxi1 transfection in Mxi1-deficient MEFs. We confirmed that ciliogenesis and Ift20 expression were regulated by Mxi1 in vitro. We also determined that Mxi1 regulates Ift20 promoter activity via Ets-1 binding to the Ift20 promoter. These results indicate that inactivating Mxi1 induces ciliary defects in polycystic kidney.
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Small heterodimer partner-targeting therapy inhibits systemic inflammatory responses through mitochondrial uncoupling protein 2.
PLoS ONE
PUBLISHED: 01-01-2013
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The orphan nuclear receptor, small heterodimer partner (SHP), appears to play a negative regulatory role in innate immune signaling. Emerging evidence warrants further study on the therapeutic targeting of SHP to suppress excessive and deleterious inflammation. Here we show that fenofibrate, which targets SHP, is required for inhibiting systemic inflammation via mitochondrial uncoupling protein 2 (UCP2). In vivo administration of fenofibrate ameliorated systemic inflammatory responses and increased survival upon experimental sepsis through SHP. An abundance of SHP was observed in mice fed fenofibrate and in cultured macrophages through LKB1-dependent activation of the AMP-activated protein kinase pathway. Fenofibrate significantly blocked endotoxin-triggered inflammatory signaling responses via SHP, but not via peroxisome proliferator-activated receptor (PPAR)-?. In addition to the known mechanism by which SHP modulates innate signaling, we identify a new role of fenofibrate-induced SHP on UCP2 induction, which is required for the suppression of inflammatory responses through modulation of mitochondrial ROS production. These data strongly suggest that the SHP-inducing drug fenofibrate paves the way for novel therapies for systemic inflammation by targeting SHP.
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The role of peroxidases in the pathogenesis of atherosclerosis.
BMB Rep
PUBLISHED: 08-30-2011
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Reactive oxygen species (ROS), which include superoxide anions and peroxides, induce oxidative stress, contributing to the initiation and progression of cardiovascular diseases involving atherosclerosis. The endogenous and exogenous factors hypercholesterolemia, hyperglycemia, hypertension, and shear stress induce various enzyme systems such as nicotinamide adenine dinucleotide (phosphate) oxidase, xanthine oxidase, and lipoxygenase in vascular and immune cells, which generate ROS. Besides inducing oxidative stress, ROS mediate signaling pathways involved in monocyte adhesion and infiltration, platelet activation, and smooth muscle cell migration. A number of antioxidant enzymes (e.g., superoxide dismutases, catalase, glutathione peroxidases, and peroxiredoxins) regulate ROS in vascular and immune cells. Atherosclerosis results from a local imbalance between ROS production and these antioxidant enzymes. In this review, we will discuss 1) oxidative stress and atherosclerosis, 2) ROS-dependent atherogenic signaling in endothelial cells, macrophages, and vascular smooth muscle cells, 3) roles of peroxidases in atherosclerosis, and 4) antioxidant drugs and therapeutic perspectives.
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Peroxiredoxin 2 deficiency exacerbates atherosclerosis in apolipoprotein E-deficient mice.
Circ. Res.
PUBLISHED: 08-11-2011
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Peroxiredoxin 2 (Prdx2), a thiol-specific peroxidase, has been reported to regulate proinflammatory responses, vascular remodeling, and global oxidative stress.
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5-(4-Hydroxy-2,3,5-trimethylbenzylidene) thiazolidine-2,4-dione attenuates atherosclerosis possibly by reducing monocyte recruitment to the lesion.
Exp. Mol. Med.
PUBLISHED: 06-22-2011
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A variety of benzylidenethiazole analogs have been demonstrated to inhibit 5-lipoxygenase (5-LOX). Here we report the anti-atherogenic potential of 5-(4-hydroxy- 2,3,5-trimethylbenzylidene) thiazolidin-2,4-dione (HMB-TZD), a benzylidenethiazole analog, and its potential mechanism of action in LDL receptor-deficient (Ldlr-/-) mice. HMB-TZD Treatment reduced leukotriene B4 (LTB4) production significantly in RAW264.7 macrophages and SVEC4-10 endothelial cells. Macrophages or endothelial cells pre-incubated with HMB-TZD for 2 h and then stimulated with lipopolysaccharide or tumor necrosis factor-alpha (TNF-?) displayed reduced cytokine production. Also, HMB-TZD reduced cell migration and adhesion in accordance with decreased proinflammatory molecule production in vitro and ex vivo. HMB-TZD treatment of 8-week-old male Ldlr-/- mice resulted in significantly reduced atherosclerotic lesions without a change to plasma lipid profiles. Moreover, aortic expression of pro-atherogenic molecules involved in the recruitment of monocytes to the aortic wall, including TNF-? , MCP-1, and VCAM-1, was downregulated. HMB-TZD also reduced macrophage infiltration into atherosclerotic lesions. In conclusion, HMB-TZD ameliorates atherosclerotic lesion formation possibly by reducing the expression of proinflammatory molecules and monocyte/macrophage recruitment to the lesion. These results suggest that HMB-TZD, and benzylidenethiazole analogs in general, may have therapeutic potential as treatments for atherosclerosis.
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Molecular targeting of atherosclerotic plaques by a stabilin-2-specific peptide ligand.
J Control Release
PUBLISHED: 03-03-2011
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Many cells, including macrophages, accumulate in atherosclerotic lesions, destabilizing plaques and driving plaque disruption. Therefore, macrophages serve as useful targets for atherosclerosis treatment and imaging. Stabilin-2 is a transmembrane protein expressed predominantly in macrophages and endothelial cells. In the present study, we found that stabilin-2 was widely expressed in atherosclerotic plaques than in normal vessel walls, and was present not only in macrophages but also in endothelial and smooth muscle cells in plaques. We used phage display technology to identify peptides that specifically bound to stabilin-2. After four rounds of selection, the most commonly isolated peptide had the sequence CRTLTVRKC, and was named S2P. We confirmed that this peptide specifically bound to stabilin-2-expressing cells in vitro and sinus endothelial cells in the spleen and lymph nodes in vivo. A FITC-conjugated synthetic CRTLTVRKC peptide was shown to home to atherosclerotic plaques in Ldlr-/- mice and to co-localize with endothelial cells, macrophages, and smooth muscle cells in such plaques. S2P conjugated to hydrophobically modified glycol chitosan nanoparticles was efficiently delivered to atherosclerotic plaques. These results show that the CRLTLTVRKC peptide homes to plaques by targeting stabilin-2; the peptide shows promise as a drug delivery moiety for, and an aid to molecular imaging of, atherosclerosis and other inflammatory diseases.
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Flt3 signaling-dependent dendritic cells protect against atherosclerosis.
Immunity
PUBLISHED: 02-16-2011
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Early events in atherosclerosis occur in the aortic intima and involve monocytes that become macrophages. We looked for these cells in the steady state adult mouse aorta, and surprisingly, we found a dominance of dendritic cells (DCs) in the intima. In contrast to aortic adventitial macrophages, CD11c(+)MHC II(hi) DCs were poorly phagocytic but were immune stimulatory. DCs were of two types primarily: classical Flt3-Flt3L signaling-dependent, CD103(+)CD11b(-) DCs and macrophage-colony stimulating factor (M-CSF)-dependent, CD14(+)CD11b(+)DC-SIGN(+) monocyte-derived DCs. Both types expanded during atherosclerosis. By crossing Flt3(-/-) to Ldlr(-/-) atherosclerosis-prone mice, we developed a selective and marked deficiency of classical CD103(+) aortic DCs, and they were associated with exacerbated atherosclerosis without alterations in blood lipids. Concomitantly, the Flt3(-/-)Ldlr(-/-) mice had fewer Foxp3(+) Treg cells and increased inflammatory cytokine mRNAs in the aorta. Therefore, functional DCs are dominant in normal aortic intima and, in contrast to macrophages, CD103(+) classical DCs are associated with atherosclerosis protection.
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Peroxisome proliferator-activated receptor {alpha} is responsible for the up-regulation of hepatic glucose-6-phosphatase gene expression in fasting and db/db Mice.
J. Biol. Chem.
PUBLISHED: 11-16-2010
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Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor ? (PPAR?) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPAR? up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPAR? up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPAR?-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPAR? binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPAR? is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.
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Nek2 and its substrate, centrobin/Nip2, are required for proper meiotic spindle formation of the mouse oocytes.
Zygote
PUBLISHED: 06-23-2010
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A typical centrosome consists of a pair of centrioles embedded in a proteinous matrix called pericentriolar material. However, the centrosomes in the mouse oocytes and early embryos lack centrioles, but consist of the ?-tubulin-enriched vesicle aggregates. We previously revealed that Nek2 and centrobin/Nip2, a centrosomal substrate of Nek2, is critical for the mouse early embryogenesis, especially at the step of spindle assembly during mitosis. In order to expand our understanding of the biological functions of Nek2, we examined expression and knockdown phenotypes of Nek2 and its substrates, centrobin and C-Nap1, in the mouse oocyte. Nek2, centrobin and C-Nap1 in the mouse oocytes were also centrosomal. Suppression of Nek2 and its substrates did not affect meiotic resumption of the oocytes. However, meiosis of the Nek2- and centrobin-suppressed oocytes was not completed, but arrested with defects in spindle assembly. No visible phenotype was observed in the C-Nap1-suppressed oocytes. These results indicate that Nek2 is critical for proper assembly of the meiotic spindles. Centrobin may be a possible substrate of Nek2 responsible for the meiotic spindle assembly in the mouse oocytes.
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Arrest defective 1 autoacetylation is a critical step in its ability to stimulate cancer cell proliferation.
Cancer Res.
PUBLISHED: 05-25-2010
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The N-acetyltransferase arrest defective 1 (ARD1) is an important regulator of cell growth and differentiation that has emerged recently as a critical molecule in cancer progression. However, the regulation of the enzymatic and biological activities of human ARD1 (hARD1) in cancer is presently poorly understood. Here, we report that hARD1 undergoes autoacetylation and that this modification is essential for its functional activation. Using liquid chromatography-tandem mass spectrometry and site-directed mutational analyses, we identified K136 residue as an autoacetylation target site. K136R mutation abolished the ability of hARD1 to promote cancer cell growth in vitro and tumor xenograft growth in vivo. Mechanistic investigations revealed that hARD1 autoacetylation stimulated cyclin D1 expression through activation of the transcription factors beta-catenin and activator protein-1. Our results show that hARD1 autoacetylation is critical for its activation and its ability to stimulate cancer cell proliferation and tumorigenesis.
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Anti-atherogenic effect of BHB-TZD having inhibitory activities on cyclooxygenase and 5-lipoxygenase in hyperlipidemic mice.
Atherosclerosis
PUBLISHED: 04-29-2010
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Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX), which play pivotal roles in atherogenesis, have been reported to be involved in plaque stability. Licofelone, a dual COX and 5-LOX inhibitor, has been reported to possess anti-atherogenic effect in rabbit atherosclerosis model. We therefore investigated the anti-atherogenic effect of BHB-TZD [5-(3,5-di-tert-butyl-4-hydroxybenzylidene)thiazolidin-2,4-dione], a dual COX and 5-LOX inhibitor, in low density lipoprotein receptor null (LDLR-/-) mice. Fifteen LDLR-/- mice were fed a western diet (control group), whereas 15 were fed a western diet plus 0.1% (w/w) BHB-TZD (BHB-TZD group). After 8 weeks, the BHB-TZD group had markedly lower serum levels of leukotriene B(4) and prostaglandin E(2) than the control group. Interestingly, BHB-TZD treatment also reduced plasma triglyceride level without significant changes in total cholesterol and HDL levels. Compared with control mice, BHB-TZD fed mice had 52% fewer fatty streak lesions in the aortic sinus, as well as fewer initial lesions in the aortic arch. Macrophage infiltration into the lesions was 40% lower, and collagen and smooth muscle cells were increased by 102% and 96%, respectively, in the BHB-TZD group compared with the control group. In addition, aortic expression of proatherogenic molecules including TNF-alpha, IL-1beta, IL-6, MCP-1 and VCAM-1, was lower in the BHB-TZD group than the control group. BHB-TZD treatment also reduced MMP-2 and MMP-9 expressions in aorta. In conclusion, BHB-TZD effectively attenuated atherosclerosis in mouse model, suggesting its therapeutic potential for atherosclerosis.
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Roles of arrest-defective protein 1(225) and hypoxia-inducible factor 1alpha in tumor growth and metastasis.
J. Natl. Cancer Inst.
PUBLISHED: 03-01-2010
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Vascular endothelial growth factor A (VEGFA), a critical mediator of tumor angiogenesis, is a well-characterized target of hypoxia-inducible factor 1 (HIF-1). Murine arrest-defective protein 1A (mARD1A(225)) acetylates HIF-1alpha, triggering its degradation, and thus may play a role in decreased expression of VEGFA.
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CD137 (4-1BB) deficiency reduces atherosclerosis in hyperlipidemic mice.
Circulation
PUBLISHED: 02-22-2010
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The tumor necrosis factor receptor superfamily, which includes CD40, LIGHT, and OX40, plays important roles in atherosclerosis. CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, has been reported to be expressed in human atherosclerotic lesions. However, limited information is available on the precise role of CD137 in atherosclerosis and the effects of blocking CD137/CD137 ligand signaling on lesion formation.
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Glutathione peroxidase 1 protects mitochondria against hypoxia/reoxygenation damage in mouse hearts.
Pflugers Arch.
PUBLISHED: 01-22-2010
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Glutathione peroxidase 1 (GPx1) plays an important role in preventing cardiac dysfunction following ischemia-reperfusion injury. However, its role in protecting cardiac mitochondria against reoxygenation-induced reactive oxygen species (ROS) generation in vivo is unclear. We examined the role of GPx1 in protecting cardiac mitochondria against hypoxia-reoxygenation (HR) damage by testing for alterations in cardiac mitochondrial function. We used a two-dimensional gel electrophoresis proteomics analysis to examine the effects of reoxygenation on cardiac protein in wild-type (GPx1(+/+)) and GPx1 knockout (GPx1(-/-)) mouse hearts. We identified 42 protein spots showing differential expression in the two groups. Sixteen of the proteins identified were located in mitochondria and were involved in a number of key metabolic pathways. To verify our proteomics findings functionally, we performed NADH autofluorescence measurements and ATP production assays. The reduced expression of oxidative phosphorylation proteins in GPx1(-/-) mice following HR treatment resulted in loss of the mitochondrial membrane potential and decreased mitochondrial respiration. Mitochondrial ROS production and oxidative mtDNA damage were increased markedly during reoxygenation in GPx1(-/-) hearts. We also found morphological abnormalities in cardiac mitochondria and myocytes in HR-treated GPx1(-/-). This is the first report of the role of GPx1 in protecting cardiac mitochondria against reoxygenation damage in vivo. These findings will help clarify the mechanisms of HR injury and will aid in the development of antioxidant therapies to prevent cardiac mitochondrial dysfunction associated with reoxygenation.
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Compensatory induction of the TRPV6 channel in a calbindin-D9k knockout mouse: Its regulation by 1,25-hydroxyvitamin D3.
J. Cell. Biochem.
PUBLISHED: 09-25-2009
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Active calcium transport is carried out by calcium channel proteins, cytosolic buffering or transfer proteins, and pump proteins. Several components of this transport system have recently been verified using gene knockout (KO) models. We previously generated calbindin-D9k (CaBP-9k) KO mice and reported that induction of expression of some calcium transport proteins can compensate for the CaBP-9k gene deficiency. In the current study, we have further clarified the compensatory regulation of calcium transport genes by two calcium regulating hormones, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and parathyroid hormone (PTH), in CaBP-9k KO mice, because the levels of these hormones differ between the KO and wild-type (WT) mice. The induction of transient receptor potential cation channel, subfamily V, member 6 (TRPV6) in the duodenum was observed in adult KO male mice but induction was not modified by physiologic doses of 1,25(OH)(2)D(3). Duodenal TRPV6 transcription in WT and female KO mice were modulated by 1,25(OH)(2)D(3) in a dose-dependent manner. This compensatory gene induction was not detected in the mice fed a vitamin D(3)-deficient diet. Compensatory gene induction was not affected by PTH. Thus, the compensatory expression of duodenal TRPV6 in the KO male mice may be tightly correlated with serum 1,25(OH)(2)D(3). Vitamin D receptor (VDR) transcription and protein levels were measured to examine whether VDR expression mediates differential regulation of duodenal TRPV6 between WT and KO mice, but expression and levels of VDR were similar in both genotypes. The compensatory TRPV6 transcripts in KO mice may be modulated by endogenous vitamin D(3) via other factors of VDR signaling complexes.
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Dietary calcium and 1,25-dihydroxyvitamin D3 regulate transcription of calcium transporter genes in calbindin-D9k knockout mice.
J. Reprod. Dev.
PUBLISHED: 07-31-2009
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The effect(s) of oral calcium and vitamin D(3) were examined on the expression of duodenal and renal active calcium transport genes, i.e., calbindin-D9k (CaBP-9k) and calbindin-D28k (CaBP-28k), transient receptor potential cation channels (TRPV5 and TRPV6), Na(+)/Ca(2+) exchanger 1 (NCX1) and plasma membrane calcium ATPase 1b (PMCA1b), in CaBP-9k KO mice. Wild-type (WT) and KO mice were provided with calcium and vitamin D(3)-deficient diets for 10 weeks. The deficient diet significantly decreased body weights compared with the normal diet groups. The serum calcium concentration of the WT mice was decreased by the deficient diet but was unchanged in the KO mice. The deficient diet significantly increased duodenal transcription of CaBP-9k and TRPV6 in the WT mice, but no alteration was observed in the KO mice. In the kidney, the deficient diet significantly increased renal transcripts of CaBP-9k, TRPV6, PMCA1b, CaBP-28k and TRPV5 in the WT mice but did not alter calcium-relating genes in the KO mice. Two potential mediators of calcium-processing genes, vitamin D receptor (VDR) and parathyroid hormone receptor (PTHR), have been suggested to be useful for elucidating these differential regulations in the calcium-related genes of the KO mice. Expression of VDR was not significantly affected by diet or the KO mutation. Renal PTHR mRNA levels were reduced by the diet, and reduced expression was also seen in the KO mice given the normal diet. Taken together, these results suggest that the active calcium transporting genes in KO mice may have resistance to the deficiency diet of calcium and vitamin D(3).
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Fenofibrate differentially regulates plasminogen activator inhibitor-1 gene expression via adenosine monophosphate-activated protein kinase-dependent induction of orphan nuclear receptor small heterodimer partner.
Hepatology
PUBLISHED: 07-14-2009
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Plasminogen activator inhibitor type I (PAI-1) is a marker of the fibrinolytic system and serves as a possible predictor for hepatic metabolic syndromes. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, is a drug used for treatment of hyperlipidemia. Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of crucial genes involved in various metabolic pathways. In this study, we show that fenofibrate increased SHP gene expression in cultured liver cells and in the normal and diabetic mouse liver by activating the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway in a PPARalpha-independent manner. Administration of transforming growth factor beta (TGF-beta) or a methionine-deficient and choline-deficient (MCD) diet to induce the progressive fibrosing steatohepatitis model in C57BL/6 mice was significantly reversed by fenofibrate via AMPK-mediated induction of SHP gene expression with a dramatic decrease in PAI-1 messenger RNA (mRNA) and protein expression along with other fibrotic marker genes. No reversal was observed in SHP null mice treated with fenofibrate. Treatment with another PPARalpha agonist, WY14643, showed contrasting effects on these marker gene expressions in wild-type and SHP null mice, demonstrating the specificity of fenofibrate in activating AMPK signaling. Fenofibrate exhibited a differential inhibitory pattern on PAI-1 gene expression depending on the transcription factors inhibited by SHP. Conclusion: By demonstrating that a PPARalpha-independent fenofibrate-AMPK-SHP regulatory cascade can play a key role in PAI-1 gene down-regulation and reversal of fibrosis, our study suggests that various AMPK activators regulating SHP might provide a novel pharmacologic option in ameliorating hepatic metabolic syndromes.
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Cyst formation in kidney via B-Raf signaling in the PKD2 transgenic mice.
J. Biol. Chem.
PUBLISHED: 05-05-2009
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The pathogenic mechanisms of human autosomal dominant polycystic kidney disease (ADPKD) have been well known to include the mutational inactivation of PKD2. Although haploinsufficiency and loss of heterozygosity at the Pkd2 locus can cause cyst formation in mice, polycystin-2 is frequently expressed in the renal cyst of human ADPKD, raising the possibility that deregulated activation of PKD2 may be associated with the cystogenesis of human ADPKD. To determine whether increased PKD2 expression is physiologically pathogenic, we generated PKD2-overexpressing transgenic mice. These mice developed typical renal cysts and an increase of proliferation and apoptosis, which are reflective of the human ADPKD phenotype. These manifestations were first observed at six months, and progressed with age. In addition, we found that ERK activation was induced by PKD2 overexpression via B-Raf signaling, providing a possible molecular mechanism of cystogenesis. In PKD2 transgenic mice, B-Raf/MEK/ERK sequential signaling was up-regulated. Additionally, the transgenic human polycystin-2 partially rescues the lethality of Pkd2 knock-out mice and therefore demonstrates that the transgene generated a functional product. Functional strengthening or deregulated activation of PKD2 may be a direct cause of ADPKD. The present study provides evidence for an in vivo role of overexpressed PKD2 in cyst formation. This transgenic mouse model should provide new insights into the pathogenic mechanism of human ADPKD.
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Decreased brain edema after collagenase-induced intracerebral hemorrhage in mice lacking the inducible nitric oxide synthase gene. Laboratory investigation.
J. Neurosurg.
PUBLISHED: 04-21-2009
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Hematoma size and brain edema after intracerebral hemorrhage (ICH) are important prognostic factors. Inducible nitric oxide synthase (iNOS) is induced after cerebral ischemia and is known to be involved in secondary neuronal injury, but its significance in ICH is unknown. The authors tested whether iNOS would influence hematoma size and brain edema after ICH.
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RhoGDI2 expression is associated with tumor growth and malignant progression of gastric cancer.
Clin. Cancer Res.
PUBLISHED: 04-07-2009
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Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a regulator of Rho family GTPase. However, there is currently no direct evidence suggesting whether RhoGDI2 activates or inhibits Rho family GTPase in vivo (and which type), and the role of RhoGDI2 in tumor remains controversial. Here, we assessed the effects of RhoGDI2 expression on gastric tumor growth and metastasis progression.
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Dexamethasone differentially regulates renal and duodenal calcium-processing genes in calbindin-D9k and -D28k knockout mice.
Exp. Physiol.
PUBLISHED: 04-04-2009
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Glucocorticoids (GCs) appear to downregulate active calcium-transporting genes in the duodenum, resulting in GC-induced calcium-absorbing disorder. In this study, we examined the effects of GCs on calcium-processing genes in the duodenum and kidney and the compensatory mechanism in calbindin-D9k (CaBP-9k) and calbindin-D28k (CaBP-28k) knockout (KO) mice. In the duodenum, we observed compensatory increases in transient receptor potential vanilloid 6 (TRPV6) mRNAs in both calbindin KO mice and CaBP-9k transcripts in CaBP-28k KO mice, and their expressions were decreased by addition of a synthetic GC, dexamethasone (Dex, 10 mg kg(-1)). In addition, the expression of plasma membrane calcium ATPase 1b (PMCA1b) underwent a compensatory increase in CaBP-9k KO mice, and was blocked by Dex, while the mRNA level of duodenal sodium-calcium exchanger 1 was not altered by KO status or Dex. The renal transcriptional levels of TRPV5 in CaBP-9k KO and CaBP-9k in CaBP-28k KO mice were upregulated in a compensatory manner, while the TRPV6 gene was downregulated following treatment with Dex in the kidney of CaBP-28k KO mice. The immunological location of these duodenal proteins as a primary target of Dex-involved regulation was not altered by Dex or KO status. To elucidate potential mechanism(s) of Dex-induced compensatory gene expression, the levels of GC receptor (GR), vitamin D receptor (VDR) and parathyroid hormone receptor (PTHR) mRNA was also measured in these tissues. Duodenal VDR transcripts were induced in a compensatory manner in both types of KO mice, and were decreased by Dex. In addition, serum corticosterone levels in both KO mice were lower than in wild-type mice. In conclusion, these results suggest that duodenal TRPV6 and CaBP-9k genes appear to be a primary target for GC-induced calcium-absorbing disorder, through direct regulation of duodenal VDR transcription.
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Characterization of ASC-2 as an antiatherogenic transcriptional coactivator of liver X receptors in macrophages.
Mol. Endocrinol.
PUBLISHED: 04-02-2009
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Activating signal cointegrator-2 (ASC-2) functions as a transcriptional coactivator of many nuclear receptors and also plays important roles in the physiology of the liver and pancreas by interacting with liver X receptors (LXRs), which antagonize the development of atherosclerosis. This study was undertaken to establish the specific function of ASC-2 in macrophages and atherogenesis. Intriguingly, ASC-2 was more highly expressed in macrophages than in the liver and pancreas. To inhibit LXR-specific activity of ASC-2, we used DN2, which contains the C-terminal LXXLL motif of ASC-2 and thereby acts as an LXR-specific, dominant-negative mutant of ASC-2. In DN2-overexpressing transgenic macrophages, cellular cholesterol content was higher and cholesterol efflux lower than in control macrophages. DN2 reduced LXR ligand-dependent increases in the levels of ABCA1, ABCG1, and apolipoprotein E (apoE) transcripts as well as the activity of luciferase reporters driven by the LXR response elements (LXREs) of ABCA1, ABCG1, and apoE genes. These inhibitory effects of DN2 were reversed by overexpression of ASC-2. Chromatin immunoprecipitation analysis demonstrated that ASC-2 was recruited to the LXREs of the ABCA1, ABCG1, and apoE genes in a ligand-dependent manner and that DN2 interfered with the recruitment of ASC-2 to these LXREs. Furthermore, low-density lipoprotein receptor (LDLR)-null mice receiving bone marrow transplantation from DN2-transgenic mice showed accelerated atherogenesis when administered a high-fat diet. Taken together, these results indicate that suppression of the LXR-specific activity of ASC-2 results in both defective cholesterol metabolism in macrophages and accelerated atherogenesis, suggesting that ASC-2 is an antiatherogenic coactivator of LXRs in macrophages.
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Signal crosstalk between estrogen and peroxisome proliferator-activated receptor alpha on adiposity.
BMB Rep
PUBLISHED: 03-03-2009
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Peroxisome proliferator-activated receptor alpha and estrogen are believed to be involved in metabolic changes leading to obesity. To test this relationship, we divided female wildtype and PPAR alpha-deficient mice fed on a high fat diet into the following groups: mock-operated, ovariectomized (OVX), and E(2)-treated. The visceral white adipose tissue and plasma cholesterol levels were increased significantly in wild type OVX and decreased in the E(2)-treated group, but interestingly not in PPAR alpha-deficient mice. The mRNA levels of lipoprotein lipase in adipose tissue were also increased in only wild type OVX and decreased significantly in E(2)-treated mice. These novel results suggest the possibility of signaling crosstalk between PPAR alpha and E2, causing obesity in vivo.
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Nuclear factor kappaB-mediated down-regulation of adhesion molecules: possible mechanism for inhibitory activity of bigelovin against inflammatory monocytes adhesion to endothelial cells.
J Ethnopharmacol
PUBLISHED: 02-25-2009
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The flowers of Inula britannica L. var. chinensis (Rupr.) Reg. (Compositae) are used in traditional medicine to treat asthma, chronic bronchitis, and acute pleurisy in China and Korea. However, the pharmacological actions of Inula britannica L. var. chinensis on endothelial cells and inflammatory monocytes are not clear. In this study, we investigated whether bigelovin, a sesquiterpene lactone isolated from the flowers of Inula britannica L. var. chinensis, inhibits monocyte adhesion and adhesion molecule expression in brain endothelial cells. We measured tumor necrosis factor-alpha (TNF-alpha)-enhanced Raw264.7 monocyte binding to brain endothelial cells and the levels of cell adhesion molecules, including vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and endothelial-selectin (E-selectin) on the surface of brain endothelial cells. Bigelovin significantly inhibited these in a dose-dependent manner without affecting cell viability. Furthermore, bigelovin suppressed the nuclear factor kappaB (NF-kappaB) promoter-driven luciferase activity, NF-kappaB activation, and degradation of NF-kappaB inhibitor protein alpha (IkappaBalpha). These results indicate that bigelovin inhibits inflammatory monocyte adhesion to endothelial cells and the expression of VCAM-1, ICAM-1, and E-selectin by blocking IkappaBalpha degradation and NF-kappaB activation.
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Effect of dietary calcium and 1,25-(OH)2D3 on the expression of calcium transport genes in calbindin-D9k and -D28k double knockout mice.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-14-2009
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The phenotypes of calbindin-D9k (CaBP-9k) and -28k (CaBP-28k) single knockout (KO) mice are similar to wild-type (WT) mice due to the compensatory action of other calcium transport proteins. In this study, we generated CaBP-9k/CaBP-28k double knockout (DKO) mice in order to investigate the importance of CaBP-9k and CaBP-28k in active calcium processing. Under normal dietary conditions, DKO mice did not exhibit any changes in phenotype or the expression of active calcium transport genes as compared to WT or CaBP-28k KO mice. Under calcium-deficient dietary conditions, the phenotype and expression of calcium transport genes in CaBP-28k KO mice were similar to WT, whereas in DKO mice, serum calcium levels and bone length were decreased. The intestinal and renal expression of transient receptor potential vanilloid member 6 (TRPV6) mRNA was significantly decreased in DKO mice fed a calcium-deficient diet as compared to CaBP-28k KO or WT mice, and DKO mice died after 4 weeks on a calcium-deficient diet. Body weight, bone mineral density (BMD) and bone length were significantly reduced in all mice fed a calcium and 1,25-(OH)(2)D(3)-deficient diet, as compared to a normal diet, and none of the mice survived more than 4 weeks. These results indicate that deletion of CaBP-28k alone does not affect body calcium homeostasis, but that deletion of CaBP-9k and CaBP-28k has a significant effect on calcium processing under calcium-deficient conditions, confirming the importance of dietary calcium and 1,25-(OH)(2)D(3) during growth and development.
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Berberine improves lipid dysregulation in obesity by controlling central and peripheral AMPK activity.
Am. J. Physiol. Endocrinol. Metab.
PUBLISHED: 01-27-2009
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AMP-activated protein kinase (AMPK) plays an important role in regulating whole body energy homeostasis. Recently, it has been demonstrated that berberine (BBR) exerts antiobesity and antidiabetic effects in obese and diabetic rodent models through the activation of AMPK in peripheral tissues. Here we show that BBR improves lipid dysregulation and fatty liver in obese mice through central and peripheral actions. In obese db/db and ob/ob mice, BBR treatment reduced liver weight, hepatic and plasma triglyceride, and cholesterol contents. In the liver and muscle of db/db mice, BBR promoted AMPK activity and fatty acid oxidation and changed expression of genes involved in lipid metabolism. Additionally, intracerebroventricular administration of BBR decreased the level of malonyl-CoA and stimulated the expression of fatty acid oxidation genes in skeletal muscle. Together, these data suggest that BBR would improve fatty liver in obese subjects, which is probably mediated not only by peripheral AMPK activation but also by neural signaling from the central nervous system.
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Evaluation of VCAM-1 antibodies as therapeutic agent for atherosclerosis in apolipoprotein E-deficient mice.
Atherosclerosis
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Blocking agents targeting cell adhesion molecules have been developed to prevent cardiovascular diseases such as atherosclerosis, whereas relatively little attention has been paid to the therapeutic potential of vascular cell adhesion molecule (VCAM)-1 as an inflammatory disease target. Two novel, fully human antibodies, H6 and 7H, against human VCAM-1 (hVCAM-1) were developed and tested to validate the hypothesis that blocking VCAM-1 ameliorates atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice.
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Ginseng Berry Extract Prevents Atherogenesis via Anti-Inflammatory Action by Upregulating Phase II Gene Expression.
Evid Based Complement Alternat Med
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Ginseng berry possesses higher ginsenoside content than its root, which has been traditionally used in herbal medicine for many human diseases, including atherosclerosis. We here examined the antiatherogenic effects of the Korean ginseng berry extract (KGBE) and investigated its underlying mechanism of action in vitro and in vivo. Administration of KGBE decreased atherosclerotic lesions, which was inversely correlated with the expression levels of phase II genes to include heme oxygenase-1 (HO-1) and glutamine-cysteine ligase (GCL). Furthermore, KGBE administration suppressed NF-?B-mediated expression of atherogenic inflammatory genes (TNF-?, IL-1?, iNOS, COX-2, ICAM-1, and VCAM-1), without altering serum cholesterol levels, in ApoE(-/-) mice fed a high fat-diet. Treatment with KGBE increased phase II gene expression and suppressed lipopolysaccharide-induced reactive oxygen species production, NF-?B activation, and inflammatory gene expression in primary macrophages. Importantly, these cellular events were blocked by selective inhibitors of HO-1 and GCL. In addition, these inhibitors reversed the suppressive effect of KGBE on TNF-?-mediated induction of ICAM-1 and VCAM-1, resulting in decreased interaction between endothelial cells and monocytes. These results suggest that KGBE ameliorates atherosclerosis by inhibiting NF-?B-mediated expression of atherogenic genes via upregulation of phase II enzymes and thus has therapeutic or preventive potential for atherosclerosis.
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Peptidomimetic small-molecule compounds promoting cardiogenesis of stem cells.
Arch. Pharm. Res.
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Embryonic stem (ES) cells may be used as an alternative source of functionally intact cardiomyocytes for ischemic heart disease. Several natural and synthetic small molecules have been identified as useful tools for controlling and manipulating stem cell renewal and differentiation. Currently, there is an urgent requirement for novel small molecules that specifically induce differentiation of stem cells into cardiomyocytes. To identify compounds that promote cardiomyogenesis of stem cells, cell-based screening of a peptidomimetic small-molecule library was carried out. A series of ?-turn peptidomimetic compounds, including CW209E, increased the expression of ?-MHC promoter-driven enhanced green fluorescent protein (EGFP) and ratio of beating embryoid bodies (EBs) without inducing cytotoxicity in mouse embryonic stem cells. CW209E also increased the number of beating EBs in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Thus, this chemical compound should be useful for elucidation of the molecular pathway of cardiogenesis and generation of cardiomyocytes ex vivo, which can be further applied for experimental or clinical cell therapy for ischemic heart diseases.
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Rho-associated coiled-coil-containing kinase 2 deficiency in bone marrow-derived cells leads to increased cholesterol efflux and decreased atherosclerosis.
Circulation
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Macrophages play a central role in the development of atherosclerosis. However, the signaling pathways that regulate their function are not well understood. The Rho-associated coiled-coil-containing kinases (ROCK1 and ROCK2) are serine-threonine protein kinases that are involved in the regulation of the actin cytoskeleton. Recent studies suggest that ROCK1 in macrophages and bone marrow-derived cells mediates atherogenesis. However, a similar role for ROCK2 in the pathogenesis of atherosclerosis has not been determined.
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Macrophage inhibitory cytokine-1 stimulates proliferation of human umbilical vein endothelial cells by up-regulating cyclins D1 and E through the PI3K/Akt-, ERK-, and JNK-dependent AP-1 and E2F activation signaling pathways.
Cell. Signal.
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Macrophage inhibitory cytokine-1 (MIC-1) is highly associated with malignant human cancers and has been suggested to be involved in tumor angiogenesis. In the present study, we examined the effect of MIC-1 on endothelial cell proliferation to confirm the angiogenesis-promoting role of MIC-1. MIC-1 treatment accelerated progression of the G(1) stage in the cell cycle of human umbilical vein endothelial cells (HUVECs), leading to an increased cell proliferation rate. MIC-1 augmented the levels of cyclins D1 and E without altering the levels of cyclin-dependent kinase (CDK) inhibitors, thereby increasing protein kinase activity of CDKs and subsequent phosphorylation of the Rb protein followed by nuclear translocation of E2F. MIC-1-induced expression of cyclins D1 and E was mediated by AP-1 and E2F-1 transcription factors, and among the AP-1 members, c-Jun and JunD appeared to participate in MIC-1-dependent transcription of the cyclin D1 gene. Additionally, the PI3K/Akt, JNK, and ERK pathways were found to mediate MIC-1-induced cyclin D1 expression in HUVECs. Importantly, lung endothelial cells isolated from MIC-1 transgenic mouse displayed a higher proliferation rate and cyclin D1 and E levels relative to their wild-type counterparts. These results suggest that MIC-1 secreted from cancer cells stimulates endothelial cell proliferation by enhancing AP-1- and E2F-dependent expression of G(1) cyclins via PI3K/Akt, JNK, and ERK signaling pathways, potentially leading to enhanced tumor angiogenesis.
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Hypertension resulting from overexpression of translationally controlled tumor protein increases the severity of atherosclerosis in apolipoprotein E knock-out mice.
Transgenic Res.
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Hypertension is a well-established etiological factor for atherogenesis. We previously showed that transgenic mice overexpressing translationally controlled tumor protein (TCTP) develop systemic arterial hypertension. In this study we explored the cardiovascular effects of TCTP overexpression and possibly of the resultant hypertension on the severity of atherosclerosis in apolipoprotein E-deficient mice. Through multiple mating of TCTP-overexpressing transgenic mice (TCTP-TG) with apolipoprotein E knock-out mice (ApoE KO), we generated non-transgenic (nTG), TCTP-TG, nTG/ApoE KO and TCTP-TG/ApoE KO mice with similar genetic background. Male mice, 7-week old, were fed a lipid-enriched Western diet for 16 weeks, and blood pressure and body weight change were monitored every 2 weeks. Plasma lipid profiles and atherosclerotic lesions in aorta were quantified at the end of study. We found that blood pressure levels of TCTP-TG and TCTP-TG/ApoE KO, were similarly elevated while nTG and nTG/ApoE KO mice were normotensive. TCTP overexpression in ApoE KO mice led to significant exacerbation of atherosclerotic lesions. Feeding Western diet resulted in increases in total cholesterol, triglyceride (TG) and low density lipoprotein, and decreased high density lipoprotein (HDL) in ApoE KO mice. No significant differences were found in plasma lipid profiles of nTG/ApoE KO and TCTP-TG/ApoE KO. This study suggests that overexpression of TCTP, which induces hypertension, also accelerates the development of atherosclerotic lesion caused by high-fat and high-cholesterol diet without significantly altering plasma lipid profiles. We conclude that TCTP-induced hypertension could increase the severity of atherosclerotic lesion and suggest that inhibition of TCTP or its signaling pathways may be a potential approach to the therapy of both diseases, hypertension and atherosclerosis.
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Receptor activator of nuclear factor-?B ligand is a novel inducer of myocardial inflammation.
Cardiovasc. Res.
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Although increased levels of myocardial receptor activator of nuclear factor (NF)-?B ligand (RANKL) have been reported in heart failure, the role of this pathway in mediating activation of inflammatory pathways during myocardial remodelling is less well understood. This study sought to determine the role of myocardial RANKL in regulating cytokine expression.
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Ginkgo biloba extract (GbE) enhances the anti-atherogenic effect of cilostazol by inhibiting ROS generation.
Exp. Mol. Med.
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In this study, the synergistic effect of 6-[4-(1-cyclohexyl- 1H-tetrazol-5-yl) butoxy]-3,4-dihydro-2(1H )-quinolinone (cilostazol) and Ginkgo biloba extract (GbE) was examined in apolipoprotein E (ApoE) null mice. Co-treatment with GbE and cilostazol synergistically decreased reactive oxygen species (ROS) production in ApoE null mice fed a high-fat diet. Co-treatment resulted in a significantly decreased atherosclerotic lesion area compared to untreated ApoE mice. The inflammatory cytokines and adhesion molecules such as monocyte chemoattractant-1 (MCP-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and VCAM-1 which can initiate atherosclerosis were significantly reduced by the co-treatment of cilostazol with GbE. Further, the infiltration of macrophages into the intima was decreased by co-treatment. These results suggest that co-treatment of GbE with cilostazol has a more potent anti-atherosclerotic effect than treatment with cilostazol alone in hyperlipidemic ApoE null mice and could be a valuable therapeutic strategy for the treatment of atherosclerosis.
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Assurance of mitochondrial integrity and mammalian longevity by the p62-Keap1-Nrf2-Nqo1 cascade.
EMBO Rep.
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Sqstm1/p62 functions in the non-canonical activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, its physiological relevance is not certain. Here, we show that p62(-/-) mice exhibited an accelerated presentation of ageing phenotypes, and tissues from these mice created a pro-oxidative environment owing to compromised mitochondrial electron transport. Accordingly, mitochondrial function rapidly declined with age in p62(-/-) mice. In addition, p62 enhanced basal Nrf2 activity, conferring a higher steady-state expression of NAD(P)H dehydrogenase, quinone 1 (Nqo1) to maintain mitochondrial membrane potential and, thereby, restrict excess oxidant generation. Together, the p62-Nrf2-Nqo1 cascade functions to assure mammalian longevity by stabilizing mitochondrial integrity.
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Reactive oxygen species play a critical role in collagen-induced platelet activation via SHP-2 oxidation.
Antioxid. Redox Signal.
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Aims: The collagen-stimulated generation of reactive oxygen species (ROS) regulates signal transduction in platelets, although the mechanism is unclear. The major targets of ROS include protein tyrosine phosphatases (PTPs). ROS-mediated oxidation of the active cysteine site in PTPs abrogates PTP catalytic activity. Results: SH2 domain-containing PTP (SHP-2) is oxidized in platelets by ROS produced upon collagen stimulation. The oxidative inactivation of SHP-2 leads to the enhanced tyrosine phosphorylation of spleen tyrosine kinase (Syk), Vav1, and Brutons tyrosine kinase (Btk) in the linker for the activation of T cells (LAT) signaling complex, which promotes the tyrosine phosphorylation-mediated activation of phospholipase C?2 (PLC?2). Moreover, we found that, relative to wild-type platelets, platelets derived from glutathione peroxidase 1 (GPx1)/catalase double-deficient mice showed enhanced cellular H2O2 levels, oxidative inactivation of SHP-2, and tyrosine phosphorylation of Syk, Vav1, Btk, and PLC?2 in response to collagen, which subsequently led to increased intracellular calcium levels, degranulation, and integrin ?IIb?3 activation. Consistent with these findings, GPx1/catalase double-deficiency accelerated the thrombotic response in FeCl3-injured carotid arteries. Innovation: The present study is the first to demonstrate that SHP-2 is targeted by ROS produced in collagen-stimulated platelets, and suggests that a novel mechanism for the regulation of platelet activation by ROS is due to oxidative inactivation of SHP-2. Conclusion: We conclude that collagen-induced ROS production leads to SHP-2 oxidation, which promotes platelet activation by upregulating tyrosine phosphorylation-based signal transduction.
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