Cordycepin ameliorates cardiac hypertrophy via activating the AMPKα pathway

Increase of myocardial oxidative stress is closely related to the occurrence and development of cardiac hypertrophy. Cordycepin, also known as 3'‐deoxyadenosine, is a natural bioactive substance extracted from Cordyceps militaris (which is widely cultivated for commercial use in functional foods and medicine). Since cordycepin suppresses oxidative stress both in vitro and in vivo, we hypothesized that cordycepin would inhibit cardiac hypertrophy by blocking oxidative stress‐dependent related signalling. In our study, a mouse model of cardiac hypertrophy was induced by aortic banding (AB) surgery. Mice were intraperitoneally injected with cordycepin (20 mg/kg/d) or the same volume of vehicle 3 days after‐surgery for 4 weeks. Our data demonstrated that cordycepin prevented cardiac hypertrophy induced by AB, as assessed by haemodynamic parameters analysis and echocardiographic, histological and molecular analyses. Oxidative stress was estimated by detecting superoxide generation, superoxide dismutase (SOD) activity and malondialdehyde levels, and by detecting the protein levels of gp91^phox and SOD. Mechanistically, we found that cordycepin activated activated protein kinase α (AMPKα) signalling and attenuated oxidative stress both in vivo in cordycepin‐treated mice and in vitro in cordycepin treated cardiomyocytes. Taken together, the results suggest that cordycepin protects against post‐AB cardiac hypertrophy through activation of the AMPKα pathway, which subsequently attenuates oxidative stress.     

ZAKβ is activated by cellular compression and mediates contraction‐induced MAP kinase signaling in skeletal muscle

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction‐induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKβ is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKβ''s ability to recognize stress fibers in cells and Z‐discs in muscle fibers when mechanically perturbed. Consequently, ZAK‐deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.     

Breviscapine protects against cardiac hypertrophy through blocking PKC‐α‐dependent signaling

Breviscapine is a mixture of flavonoid glycosides extracted from the Chinese herbs. Previous studies have shown that breviscapine possesses comprehensive pharmacological functions. However, very little is known about whether breviscapine have protective role on cardiac hypertrophy. The aim of the present study was to determine whether breviscapine attenuates cardiac hypertrophy induced by angiotensin II (Ang II) in cultured neonatal rat cardiac myocytes in vitro and pressure‐overload‐induced cardiac hypertrophy in mice in vivo. Our data demonstrated that breviscapine (2.5–15 µM) dose‐dependently blocked cardiac hypertrophy induced by Ang II (1 µM) in vitro. The results further revealed that breviscapine (50 mg/kg/day) prevented cardiac hypertrophy induced by aortic banding as assessed by heart weight/body weight and lung weight/body weight ratios, echocardiographic parameters, and gene expression of hypertrophic markers. The inhibitory effect of breviscapine on cardiac hypertrophy is mediated by disrupting PKC‐α‐dependent ERK1/2 and PI3K/AKT signaling. Further studies showed that breviscapine inhibited inflammation by blocking NF‐κB signaling, and attenuated fibrosis and collagen synthesis through abrogating Smad2/3 signaling. Therefore, these findings indicate that breviscapine, which is a potentially safe and inexpensive therapy for clinical use, has protective potential in targeting cardiac hypertrophy and fibrosis through suppression of PKC‐α‐dependent signaling. J. Cell. Biochem. 109: 1158–1171, 2010. © 2010 Wiley‐Liss, Inc.     

Involvement of PPARα and PPARγ in apoptosis and proliferation of human hepatocarcinoma HepG2 cells

Peroxisome proliferator‐activated receptors (PPARs) mediate the effects of various ligands, known as peroxisome proliferators, a heterogeneous class of compounds including industrial chemicals, pharmaceuticals, and biomolecules such as fatty acids and eicosanoids. Among peroxisome proliferators, fibrate derivatives are considered specific ligands for PPARα, whereas eicosanoids, such as PGJ2, for PPARγ. The study aimed to clarify the relation between PPARs and apoptosis or proliferation on the same type of cells, using clofibrate as specific ligand of PPARα and PGJ2 as specific ligand of PPARγ. The cells used were human hepatocarcinoma HepG2 cells. The results showed that PPARα protein content increased in HepG2 cells treated with clofibrate, causing apoptosis in a time‐ and concentration‐dependent way, as evidenced by the citofluorimetric assay and determination of BAD, myc and protein phosphatase 2A protein content. It also emerged that PPARγ increased in the same cells when treated with a specific ligand of this PPAR; in this case the increase of PPARγ did not cause an increase of apoptosis, but a time‐ and concentration‐dependent inhibition of cell proliferation, evidenced by decreased cell numbers and increased number of cells in the G0/G1 phase of the cycle. It may be concluded that PPARα is chiefly related to apoptosis and PPARγ to cell proliferation. Copyright © 2010 John Wiley & Sons, Ltd.     

CYP2J2 and its metabolites (epoxyeicosatrienoic acids) attenuate cardiac hypertrophy by activating AMPKα2 and enhancing nuclear translocation of Akt1

Cytochrome P450 epoyxgenase 2J2 and epoxyeicosatrienoic acids (EETs) are known to protect against cardiac hypertrophy and heart failure, which involve the activation of 5′‐AMP‐activated protein kinase (AMPK) and Akt. Although the functional roles of AMPK and Akt are well established, the significance of cross talk between them in the development of cardiac hypertrophy and antihypertrophy of CYP2J2 and EETs remains unclear. We investigated whether CYP2J2 and its metabolites EETs protected against cardiac hypertrophy by activating AMPKα2 and Akt1. Moreover, we tested whether EETs enhanced cross talk between AMPKα2 and phosphorylated Akt1 (p‐Akt1), and stimulated nuclear translocation of p‐Akt1, to exert their antihypertrophic effects. AMPKα2^?/? mice that overexpressed CYP2J2 in heart were treated with Ang II for 2 weeks. Interestingly, overexpression of CYP2J2 suppressed cardiac hypertrophy and increased levels of atrial natriuretic peptide (ANP) in the heart tissue and plasma of wild‐type mice but not AMPKα2^?/? mice. The CYP2J2 metabolites, 11,12‐EET, activated AMPKα2 to induce nuclear translocation of p‐Akt1 selectively, which increased the production of ANP and therefore inhibited the development of cardiac hypertrophy. Furthermore, by co‐immunoprecipitation analysis, we found that AMPKα2β2γ1 and p‐Akt1 interact through the direct binding of the AMPKγ1 subunit to the Akt1 protein kinase domain. This interaction was enhanced by 11,12‐EET. Our studies reveal a novel mechanism in which CYP2J2 and EETs enhanced Akt1 nuclear translocation through interaction with AMPKα2β2γ1 and protect against cardiac hypertrophy and suggest that overexpression of CYP2J2 might have clinical potential to suppress cardiac hypertrophy and heart failure.     

LYMPHOTOXIN-α (TNF-β) DURING SEPSIS

T cell release of lymphotoxin-α (LT-α, or TNF-β) is stimulated by pyrogenic exotoxins of Gram-positive bacteria and mitogens. In contrast to TNF-α, it is unknown whether LT-α plays any role in the pathogenesis of sepsis and, in particular, the pathogenesis of Gram-positive sepsis. Sera from patients with sepsis were examined for LT-α and compared with normal volunteers and pregnant women. LT-α was detected in 33% of sepsis sera (mean 608.4 pg/ml SE 306), 16% of normal sera (mean 167 pg/ml SE 87) and 23% of sera from pregnant women (mean 714 pg/ml SE 191). These differences were not significant and there were no differences within species sera when grouped by the type of causative organism, or disease severity. LT-α detected by immunoassay in serum was not bioactive, in contrast to that produced in cell culture. Recombinant soluble TNF receptors (rSTNFR) neutralized the bioactivity of recombinant LT-α at rSTNFR concentrations which did not interfere with immunoreactivity and which are known to prevailin vivo. Hence, LT-α is unlikely to have a critical role in the pathogenesis of sepsis. Much of the potential bioactivity of this lymphokine may be abrogated by TNFR in serum.     

Characterization of mice deficient in interleukin-1β converting enzyme

Interleukin-1β converting enzyme (ICE) processes the inactive proIL-1β to the proinflammatory mature IL-1β. ICE belongs to a family of cysteine proteases that have been implicated in apoptosis. To address the biological functions of ICE, we generated ICE-deficient mice through gene targeting technology. ICE-deficient mice developed normally, appeared healthy, and were fertile. Peritoneal macrophages from ICE-deficient mice underwent apoptosis normally upon ATP treatment. Thymocytes from young ICE-deficient mice also underwent apoptosis when triggered by dexamethasone, gamma irradiation, or aging. ICE-deficient mice had a major defect in the production of mature IL-1β and had impaired IL-1α production on LPS stimulation in vitro and in vivo. ICE-deficient mice were resistant to LPS-induced endotoxic shock. J. Cell. Biochem. 64:27–32. © 1997 Wiley-Liss, Inc.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

GADD45α mediates arsenite‐induced cell apoptotic effect in human hepatoma cells via JNKs/AP‐1‐dependent pathway

Arsenite (As(III)), an effective chemotherapeutic agent for the acute promyelocytic leukemia (APL) and multiple myeloma (MM), might be also a promise for the therapy of other cancers, including the solid tumors. However, the molecular bases of arsenite‐induced cytotoxicity in the tumor cells have not been fully defined. In this study, we have disclosed that arsenite effectively induces the apoptotic response in the HepG2 human hepatoma cells by triggering GADD45α induction and the subsequent activation of JNKs/AP‐1 cell death pathway. However, signaling events relating to GADD45α/JNKs/AP‐1 pathway activation have not been observed in HL7702 human diploid hepatic cells under the same arsenite exposure condition. Our results thus have illustrated the selective pro‐apoptotic role of arsenite in the hepatoma cells by activating GADD45α‐dependent cell death pathway whereas with little effect on the normal hepatic cells. The approaches to up‐regulate GADD45α levels might be helpful in improving the chemotherapeutic action of arsenite on certain solid tumors including hepatoma. J. Cell. Biochem. 109: 1264–1273, 2010. © 2010 Wiley‐Liss, Inc.     

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

Peroxisome proliferator‐activated receptors δ (PPARδ) is known to be expressed ubiquitously, and the predominant PPAR subtype of cardiac cells. However, relatively less is known regarding the role of PPARδ in cardiac cells except that PPARδ ligand treatment protects cardiac hypertrophy by inhibiting NF‐κB activation. Thus, in the present study, we examined the effect of selective PPARδ ligand L‐165041 on angiotensin II (AngII) induced cardiac hypertrophy and its underlying mechanism using cardiomyocyte. According to our data, L‐165041 (10 µM) inhibited AngII‐induced [³H] leucine incorporation, induction of the fetal gene atrial natriuretic factor (ANF) and increase of cardiomyocyte size. Previous studies have implicated the activation of focal adhesion kinase (FAK) in the progress of cardiomyocyte hypertrophy. L‐165041 pretreatment significantly inhibited AngII‐induced intracellular Ca²⁺ increase and subsequent phosphorylation of FAK. Further experiment using Ca²⁺ ionophore A23187 confirmed that Ca²⁺ induced FAK phosphorylation, and this was also blocked by L‐165041 pretreatment. In addition, overexpression of PPARδ using adenovirus significantly inhibited AngII‐induced intracellular Ca²⁺ increase and FAK expression, while PPARδ siRNA treatment abolished the effect of L‐165041. These data indicate that PPARδ ligand L‐165041 inhibits AngII induced cardiac hypertrophy by suppressing intracellular Ca²⁺/FAK/ERK signaling pathway in a PPARδ dependent mechanism. J. Cell. Biochem. 106: 823–834, 2009. © 2009 Wiley‐Liss, Inc.     

Induction of intracellular ceramide by interleukin-1β in oligodendrocytes

The sphingomyelin pathway has been implicated in mediating the effect of several extracellular agents leading to important biochemical and cellular changes. The aim of this investigation is to study interleukin-1β (IL-1β) signaling in oligodendrocytes. For this purpose, the CG4 oligodendrocyte cells were differentiated and incubated with IL-1β. This treatment induced a time- and dose-dependent increase of the endocellular ceramide. To mimic the effect of the elevation of endogenous ceramide, the CG4 cells were treated with the ceramide analogue C2-ceramide. Cell survival, measured with the MTT assay, showed that, by increasing the concentration of ceramide, up to 40% of CG4 cells were dying within 6 h, similar data were obtained with the primary differentiated oligodendrocytes. Condensation of chromatin, nuclear fragmentation, and formation of apoptotic bodies indicated that apoptosis was the cause of death. Surprisingly, long-term exposure (72 h) to increasing concentrations of IL-1β, which increases intracellular ceramide, did not induce oligodendroglial cell death. These results show that an increase of intracellular ceramide is not sufficient to induce apoptosis in oligodendrocytes and that IL-1β signaling through the ceramide pathway in these cells can mediate functions other than programmed cell death. J. Cell Biochem. 66:532–541, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

Rac1 activation induces tumour necrosis factor‐α expression and cardiac dysfunction in endotoxemia

Induction of tumour necrosis factor‐α (TNF‐α) expression leads to myocardial depression during sepsis. However, the underlying molecular mechanisms are not fully understood. The aim of this study was to investigate the role of Rac1 in TNF‐α expression and cardiac dysfunction during endotoxemia and to determine the involvement of phosphoinositide‐3 kinase (PI3K) in lipopolysaccharide (LPS)‐induced Rac1 activation. Our results showed that LPS‐induced Rac1 activation and TNF‐α expression in cultured neonatal mouse cardiomyocytes. The response was inhibited in Rac1 deficient cardiomyocytes or by a dominant‐negative Rac1 (Rac1N17). To determine whether PI3K regulates Rac1 activation, cardiomyocytes were treated with LY294002, a PI3K selective inhibitor. Treatment with LY294002 decreased Rac1 activity as well as TNF‐α expression stimulated by LPS. Furthermore, inhibition of PI3K and Rac1 activity decreased LPS‐induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation. To investigate the role of Rac1 in myocardial depression during endotoxemia in vivo, wild‐type and cardiomyocyte‐specific Rac1 deficient mice were treated with LPS (2 mg/kg, i.p.). Deficiency in Rac1 significantly decreased myocardial TNF‐α expression and improved cardiac function during endotoxemia. We conclude that PI3K‐mediated Rac1 activation is required for induction of TNF‐α expression in cardiomyocytes and cardiac dysfunction during endotoxemia. The effect of Rac1 on TNF‐α expression seems to be mediated by increased NADPH oxidase activity and ERK1/2 phosphorylation.     

Troxerutin downregulates C/EBP‐β gene expression via modulating the IFNγ‐ERK1/2 signaling pathway to ameliorate rotenone‐induced retinal neurodegeneration

Troxerutin, a natural flavonoid guards against oxidative stress and apoptosis with a high capability of passing through the blood‐brain barrier. Our aim was to investigate the role of troxerutin in experimentally induced retinal neurodegeneration by modulating the interferon‐gamma (IFNγ)‐extracellular signal‐regulated kinases 1/2 (ERK1/2)‐CCAAT enhancer‐binding protein β (C/EBP‐β) signaling pathway. Three groups of rats (10 each group) were included. Group I (control group), group II (rotenone treated group): the rats were injected subcutaneously with a single rotenone dosage of 3 mg/kg repeated every 48 hours for 60 days to trigger retinal neurodegeneration. Group III (troxerutin‐treated group): rats received troxerutin (150 mg/kg/day) by oral gavage 1 hour before rotenone administration. A real‐time polymerase chain reaction technique was applied to measure messenger RNA (mRNA) levels of retinal C/EBP‐β. Enzyme‐linked immunosorbent assay technique was utilized to assay tumor necrosis factor‐α (TNF‐α), IFNγ, and ERK1/2 levels. Finally, reactive oxygen species (ROS), as well as carbonylated protein (CP) levels, were assessed spectrophotometrically. Improved retinal neurodegeneration by downregulation of C/EBP‐β mRNA gene expression, also caused a significant reduction of TNF‐α, IFNγ, ERK1/2 as well as ROS and CP levels compared with the diseased group. These findings could hold promise for the usage of troxerutin as a protective agent against rotenone‐induced retinal neurodegeneration.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Ginsenoside Rb1 ameliorates CKD‐associated vascular calcification by inhibiting the Wnt/β‐catenin pathway

Vascular calcification (VC) is a pathological process underpinning major cardiovascular conditions and has attracted public attention due to its high morbidity and mortality. Chronic kidney disease (CKD) is a common disease related to VC. Ginsenoside Rb1 (Rb1) has been reported to protect the cardiovascular system against vascular diseases, yet its role in VC and the underlying mechanisms remain unclear. In this study, we established a CKD‐associated VC rat model and a β‐glycerophosphate (β‐GP)‐induced vascular smooth muscle cell (VSMC) calcification model to investigate the effects of Rb1 on VC. Our results demonstrated that Rb1 ameliorated calcium deposition and VSMC osteogenic transdifferentiation both in vivo and in vitro. Rb1 treatment inhibited the Wnt/β‐catenin pathway by activating peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), and confocal microscopy was used to show that Rb1 inhibited β‐catenin nuclear translocation in VSMCs. Furthermore, SKL2001, an agonist of the Wnt/β‐catenin pathway, compromised the vascular protective effect of Rb1. GW9662, a PPAR‐γ antagonist, reversed Rb1's inhibitory effect on β‐catenin. These results indicate that Rb1 exerted anticalcific properties through PPAR‐γ/Wnt/β‐catenin axis, which provides new insights into the potential theraputics of VC.