An evolutionarily-conserved promoter allele governs HMG-CoA reductase expression in spontaneously hypertensive rat.

3-Hydroxy-3-methyl glutaryl-coenzyme A reductase (Hmgcr) encodes the rate-limiting enzyme in the cholesterol biosynthesis pathway. The regulation of Hmgcr in rat models of genetic hypertension (viz. Spontaneously Hypertensive Rat [SHR] and its normotensive control Wistar/Kyoto [WKY] strain) is unclear. Interestingly, Hmgcr transcript and protein levels are diminished in liver tissues of SHR as compared to WKY. This observation is consistent with the diminished plasma cholesterol level in SHR animals. However, the molecular basis of these apparently counter-intuitive findings remains completely unknown. Sequencing of the Hmgcr promoter in SHR and WKY strains reveals three variations: A-405G, C-62T and a 11 bp insertion (-398_-388insTGCGGTCCTCC) in SHR. Among these variations, A-405G occurs at an evolutionarily-conserved site among many mammals. Moreover, SHR-Hmgcr promoter displays lower activity than WKY-Hmgcr promoter in various cell lines. Transient transfections of Hmgcr-promoter mutants and in silico analysis suggest altered binding of Runx3 and Srebf1 across A-405G site. On the other hand, C-62T and -398_-388insTGCGGTCCTCC variations do not appear to contribute to the reduced Hmgcr promoter activity in SHR as compared to WKY. Indeed, chromatin immunoprecipitation assays confirm differential binding of Runx3 and Srebf1 to Hmgcr promoter leading to reduced expression of Hmgcr in SHR as compared to WKY under basal as well as cholesterol-modulated conditions. Taken together, this study provides, for the first time, molecular basis for diminished Hmgcr expression in SHR animals, which may account for the reduced circulating cholesterol level in this widely-studied model for cardiovascular diseases.

Hypoxia-mediated regulation of mitochondrial transcription factors in renal epithelial cells: implications for hypertensive renal physiology.

Kidneys have a high resting metabolic rate and low partial pressure of oxygen due to enhanced mitochondrial oxygen consumption and ATP production needed for active solute transport. Heightened mitochondrial activity leads to progressively increasing hypoxia from the renal cortex to the renal medulla. Renal hypoxia is prominent in hypertensive rats due to increased sodium reabsorption within the nephrons, which demands higher energy production by oxidative phosphorylation (OXPHOS). Consequently, spontaneously hypertensive rats (SHR) display greater oxygen deficiency (hypoxia) than normotensive Wistar Kyoto rats (WKY). Here, we sought to investigate the expression of key proteins for mitochondrial biogenesis in SHR and WKY, and study the regulation of mitochondrial transcription factors (mtTFs) under in vitro hypoxic conditions in renal epithelial cells. We report that renal expressions of hypoxia-inducible factor-1-alpha (HIF-1α), peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1α), mtTFs, and OXPHOS proteins are elevated in SHR compared to WKY. In addition, our experiments in cultured kidney cells demonstrate that acute hypoxia augments the expression of these genes. Furthermore, we show that the transcripts of HIF-1α and mtTFs are positively correlated in various human tissues. We reveal, for the first time to our knowledge, that HIF-1α transactivates mtTF genes by direct interaction with their promoters in rat kidney epithelial cells (NRK-52E) under acute hypoxia. Concomitant increases in the mitochondrial DNA and RNA, and OXPHOS proteins are observed. Taken together, this study suggests that hypoxia within the renal epithelial cells may enhance mitochondrial function to meet the energy demand in proximal tubular cells during prehypertensive stages in kidneys of young SHR.

MicroRNA 27a Is a Key Modulator of Cholesterol Biosynthesis.

Hypercholesterolemia is a strong predictor of cardiovascular diseases. The 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (Hmgcr) coding for the rate-limiting enzyme in the cholesterol biosynthesis pathway is a crucial regulator of plasma cholesterol levels. However, the posttranscriptional regulation of Hmgcr remains poorly understood. The main objective of this study was to explore the role of microRNAs (miRNAs) in the regulation of Hmgcr expression. Systematic in silico predictions and experimental analyses reveal that miRNA 27a (miR-27a) specifically interacts with the Hmgcr 3' untranslated region in murine and human hepatocytes. Moreover, our data show that Hmgcr expression is inversely correlated with miR-27a levels in various cultured cell lines and in human and rodent tissues. Actinomycin D chase assays and relevant experiments demonstrate that miR-27a regulates Hmgcr by translational attenuation followed by mRNA degradation. Early growth response 1 (Egr1) regulates miR-27a expression under basal and cholesterol-modulated conditions. miR-27a augmentation via tail vein injection of miR-27a mimic in high-cholesterol-diet-fed Apoe-/- mice shows downregulation of hepatic Hmgcr and plasma cholesterol levels. Pathway and gene expression analyses show that miR-27a also targets several other genes (apart from Hmgcr) in the cholesterol biosynthesis pathway. Taken together, miR-27a emerges as a key regulator of cholesterol biosynthesis and has therapeutic potential for the clinical management of hypercholesterolemia.

Measurement of Aortic Cell Fluid-Phase Pinocytosis in vivo by Flow Cytometry.

OBJECTIVE: Fluid-phase pinocytosis is a receptor-independent mechanism of endocytosis that occurs in all mammalian cells and may be a mechanism for the uptake of LDL by macrophages. As there are currently no methods for the measurement of fluid-phase pinocytosis by individual aortic cells in vivo, we sought to identify a suitable method. METHODS: ApoE-/- mice were retro-orbitally injected with AngioSPARK fluorescent nanoparticles specifically designed to not interact with cells. After 24 h, mice were sacrificed, and the aortas were isolated and then digested to analyze aortic cell uptake of AngioSPARK by flow cytometry. RESULTS: CD11b-expressing aortic macrophages from mice injected with AngioSPARK showed high levels of fluid-phase pinocytosis compared to aortic cells not expressing CD11b (4,393.7 vs. 408.3 mean fluorescence intensity [MFI], respectively). CONCLUSION: This new technique allows for the measurement of fluid-phase pinocytosis by aortic cells in vivo, making it possible to examine the cell-signaling molecules and drugs that affect this process. Published by S. Karger AG, Basel.

Progression and Characterization of the Accelerated Atherosclerosis in Iliac Artery of New Zealand White Rabbits: Effect of Simvastatin.

OBJECTIVE: Although atherosclerosis is described in New Zealand White rabbit's iliac artery, yet details of time-dependent atherosclerosis progression are not well known. Further, a well characterized accelerated model of atherosclerosis is also required for the screening of candidate drugs to target specific steps of atherosclerosis development. The present study extensively characterizes the time-dependent plaque composition and functional responses of the atherosclerosis in rabbit iliac artery and its modification by simvastatin. METHODS: Atherosclerosis was induced with a combination of balloon injury and atherogenic diet (AD) (1% cholesterol, 6% peanut oil) in rabbit's iliac artery. Atherosclerosis progression was evaluated on days 8, 10, 15, 21, 35, and 56 after AD feeding. The plaque characterization was done using histology, real-time reverse transcription-polymerase chain reaction, and vasoreactivity experiments. The standard anti-hyperlipidemic drug, simvastatin (5 mg·kg·d), was used to investigate its effect on atherosclerotic changes. RESULTS: Plasma lipids were elevated in a progressive manner after AD feeding from days 8 to 56. Similarly, arterial lipids, Monocyte Chemoattractant Protein-1 (MCP-1) level along with infiltration of macrophages in the lesion area were also increased from day 15 onward. This resulted in a significant increase in the plaque area and intimal-medial thickness ratio in contrast to normal animals. Inflammatory milieu was observed with a significant increase in expression of pro-inflammatory regulators like MCP-1, Tumor Necrosis Factor-α (TNF-α) and Vascular Cell Adhesion Molecule-1 (VCAM-1), whereas anti-inflammatory cytokine interleukin 10 decreased as disease progressed. Endothelial dysfunction was also observed, specifically Acetylcholine (ACh)-induced vasorelaxation was reduced from day 8 onward, whereas the phenylephrine-induced vasoconstriction response was progressively reduced from day 15 in the iliac artery. Ground substances including proteoglycans, α-actin, and collagen content along with metalloproteinase-9 and Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibitors were significantly augmented at later time points, day 21 onward. Simvastatin treatment for 35 days, at a dose having no significant effect on plasma lipid levels, significantly reduced atherosclerotic progression as evident by reduced macrophage content, inflammatory burden, and extracellular matrix component like proteoglycans and metalloproteinase-9. CONCLUSIONS: The authors observed that AD feeding with balloon injury in the rabbit iliac artery accelerated the progression of atherosclerosis and exhibited predominant features of type III human lesion within 8 weeks (56 days). Simvastatin treatment for 35 days exhibited anti-atherosclerotic efficacy without significantly lowering the circulating lipids. The current study thus provides an insight into the time-dependent atherosclerotic progression in rabbit iliac artery and highlights its utility for anti-atherosclerotic evaluation of the candidate drugs.

Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism.

Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway.

A Standardized Chemically Modified Curcuma longa Extract Modulates IRAK-MAPK Signaling in Inflammation and Potentiates Cytotoxicity.

The TLR/IL-1R pathway is a critical signaling module that is misregulated in pathologies like inflammation and cancer. Extracts from turmeric (Curcuma longa L.) enriched in curcumin and carbonyls like turmerones have been shown to exert potent anti-inflammatory effects. The present study evaluated the anti-inflammatory activity, cytotoxic effect and the underlying mechanism of a novel chemically modified, non-carbonyl compound enriched Curcuma longa L. (C. longa) extract (CMCE). CMCE (1 or 10 µg/mL; 14 h) significantly decreased LPS (50-100 ng/mL) induced TNF-α and IL-1ß production in THP-1 cells, human, and mouse whole blood as measured by ELISA. LPS-induced IRAK1, MAPK activation, TLR4 expression, TLR4-MyD88 interaction, and IκBα degradation were significantly reduced in CMCE pre-treated THP-1 cells as assessed by Western blotting. CMCE (30, 100, and 300 mg/kg; 10 days p.o.) pre-treated and LPS (10 mg/kg) challenged Swiss mice exhibited attenuated plasma TNF-α, IL-1ß, nitrite, aortic iNOS expression, and vascular dysfunction. In a PI permeability assay, cell lines derived from acute myeloid leukemia were most sensitive to the cytotoxic effects of CMCE. Analysis of Sub-G1 phase, Annexin V-PI positivity, loss of mitochondrial membrane potential, increased caspase-3, and PARP-1 activation confirmed CMCE induced apoptosis in HL-60 cells. IRAK inhibition also sensitized HL-60 cells to CMCE induced cytotoxicity. The present study defines the mechanism underlying the action of CMCE and suggests a therapeutic potential for its use in sepsis and leukemia.

The IRAK-ERK-p67phox-Nox-2 axis mediates TLR4, 2-induced ROS production for IL-1ß transcription and processing in monocytes.

In monocytic cells, Toll-like receptor 4 (TLR4)- and TLR2-induced reactive oxygen species (ROS) cause oxidative stress and inflammatory response; however, the mechanism is not well understood. The present study investigated the role of interleukin-1 receptor-associated kinase (IRAK), extracellular signal-regulated kinase (ERK), p67phox and Nox-2 in TLR4- and TLR2-induced ROS generation during interleukin-1 beta (IL-1ß) transcription, processing, and secretion. An IRAK1/4 inhibitor, U0126, PD98059, an NADPH oxidase inhibitor (diphenyleneiodonium (DPI)), and a free radical scavenger (N-acetyl cysteine (NAC))-attenuated TLR4 (lipopolysaccharide (LPS))- and TLR2 (Pam3csk4)-induced ROS generation and IL-1ß production in THP-1 and primary human monocytes. An IRAK1/4 inhibitor and siRNA-attenuated LPS- and Pam3csk4-induced ERK-IRAK1 association and ERK phosphorylation and activity. LPS and Pam3csk4 also induced IRAK1/4-, ERK- and ROS-dependent activation of activator protein-1 (AP-1), IL-1ß transcription, and IL-1ß processing because significant inhibition in AP-1 activity, IL-1ß transcription, Pro- and mature IL-ß expression, and caspase-1 activity was observed with PD98059, U0126, DPI, NAC, an IRAK1/4 inhibitor, tanshinone IIa, and IRAK1 siRNA treatment. IRAK-dependent ERK-p67phox interaction, p67phox translocation, and p67phox-Nox-2 interaction were observed. Nox-2 siRNA significantly reduced secreted IL-1ß, IL-1ß transcript, pro- and mature IL-1ß expression, and caspase-1 activity indicating a role for Nox-2 in LPS- and Pam3csk4-induced IL-1ß production, transcription, and processing. In the present study, we demonstrate that the TLR4- and TLR2-induced IRAK-ERK pathway cross-talks with p67phox-Nox-2 for ROS generation, thus regulating IL-1ß transcription and processing in monocytic cells.

L-Plastin S-glutathionylation promotes reduced binding to ß-actin and affects neutrophil functions.

Posttranslational modifications (PTMs) of cytoskeleton proteins due to oxidative stress associated with several pathological conditions often lead to alterations in cell function. The current study evaluates the effect of nitric oxide (DETA-NO)-induced oxidative stress-related S-glutathionylation of cytoskeleton proteins in human PMNs. By using in vitro and genetic approaches, we showed that S-glutathionylation of L-plastin (LPL) and ß-actin promotes reduced chemotaxis, polarization, bactericidal activity, and phagocytosis. We identified Cys-206, Cys-283, and Cys-460as S-thiolated residues in the ß-actin-binding domain of LPL, where cys-460 had the maximum score. Site-directed mutagenesis of LPL Cys-460 further confirmed the role in the redox regulation of LPL. S-Thiolation diminished binding as well as the bundling activity of LPL. The presence of S-thiolated LPL was detected in neutrophils from both diabetic patients and db/db mice with impaired PMN functions. Thus, enhanced nitroxidative stress may results in LPL S-glutathionylation leading to impaired chemotaxis, polarization, and bactericidal activity of human PMNs, providing a mechanistic basis for their impaired functions in diabetes mellitus.

Fluid-phase pinocytosis of native low density lipoprotein promotes murine M-CSF differentiated macrophage foam cell formation.

During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR-/-) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR-/- macrophages with increasing concentrations of (125)I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on (125)I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect (125)I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR-/- mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.

Cytokines induced neutrophil extracellular traps formation: implication for the inflammatory disease condition.

Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1ß, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1ß, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1ß, or IL-8 antibodies reduced the NETs release. Role of IL-1ß, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects.

Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential.

Neutrophils expel extracellular traps (NETs) to entrap and exterminate the invaded micro-organisms. Acute/chronic inflammatory disorders are often observed with aberrantly enhanced NETs formation and high nitric oxide (NO) availability. Recent study from this laboratory demonstrated release of NETs from human neutrophils following treatment with SNP or SNAP. This study is an extension of our previous finding to explore the extracellular bacterial killing, source of DNA in the expelled NETs, their ability to induce proinflammatory cytokines release from platelets/THP-1 cells, and assessment of NO-mediated free radical formation by using a consistent NO donor, DETA-NONOate. NO-mediated NETs exhibited extracellular bacterial killing as determined by colony forming units. NO-mediated NETs formation was due to the activation of NADPH oxidase and myeloperoxidase. NO- or PMA-mediated NETs were positive for both nuclear and mitochondrial DNA as well as proteolytic enzymes. Incubation of NETs with human platelets enhanced the release of IL-1ß and IL-8, while with THP-1 cells, release of IL-1ß, IL-8, and TNFα was observed. This study demonstrates that NO by augmenting enzymatic free radical generation release NETs to promote extracellular bacterial killing. These NETs were made up of mitochondrial and nuclear DNA and potentiated release of proinflammatory cytokines.

Murine bone marrow-derived macrophages differentiated with GM-CSF become foam cells by PI3Kγ-dependent fluid-phase pinocytosis of native LDL.

Accumulation of cholesterol by macrophage uptake of LDL is a key event in the formation of atherosclerotic plaques. Previous research has shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) is present in atherosclerotic plaques and promotes aortic lipid accumulation. However, it has not been determined whether murine GM-CSF-differentiated macrophages take up LDL to become foam cells. GM-CSF-differentiated macrophages from LDL receptor-null mice were incubated with LDL, resulting in massive macrophage cholesterol accumulation. Incubation of LDL receptor-null or wild-type macrophages with increasing concentrations of ¹²5I-LDL showed nonsaturable macrophage LDL uptake that was linearly related to the amount of LDL added, indicating that LDL uptake was mediated by fluid-phase pinocytosis. Previous studies suggest that phosphoinositide 3-kinases (PI3K) mediate macrophage fluid-phase pinocytosis, although the isoform mediating this process has not been determined. Because PI3Kγ is known to promote aortic lipid accumulation, we investigated its role in mediating macrophage fluid-phase pinocytosis of LDL. Wild-type macrophages incubated with LDL and the PI3Kγ inhibitor AS605240 or PI3Kγ-null macrophages incubated with LDL showed an ∼50% reduction in LDL uptake and cholesterol accumulation compared with wild-type macrophages incubated with LDL only. These results show that GM-CSF-differentiated murine macrophages become foam cells by fluid-phase pinocytosis of LDL and identify PI3Kγ as contributing to this process.

Increased myeloperoxidase enzyme activity in plasma is an indicator of inflammation and onset of sepsis.

INTRODUCTION: Circulating lipopolysaccharides released from bacteria may activate both neutrophils and monocytes. The activated neutrophils release myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to evaluate MPO enzyme activity in plasma of critically ill patients and to check the hypothesis that these concentrations in plasma would be higher in sepsis and systemic inflammatory conditions, as neutrophils release their contents before proliferating in response to stress. MATERIAL AND METHODS: Blood samples were collected from 105 critically ill patients admitted to the intensive care unit, consisting of those with systemic inflammatory response syndrome (n = 42), sepsis (n = 37), and septic shock (n = 26). Plasma MPO enzyme activity was determined by o-dianisidine-H(2)O(2) method, modified for 96-well plates. RESULTS: The plasma MPO enzyme activity in sepsis patients was significantly higher than that in the control group (mean, 2.4 ± 1.8 in sepsis and 1.86 ± 1.2 nmol per milligram protein per 10 minutes in systemic inflammatory response syndrome vs 0.32 ± 0.11 nmol per milligram protein per 10 minutes in healthy controls). Mean plasma lactate levels in sepsis (7.8 ± 1.2 mmol/L) and shock patients (9.5 ± 1.2 mmol/L) and cytokines like tumor necrosis factor-α, interleukin-8, and interleukin-1ß were simultaneously evaluated to establish onset of inflammation and sepsis. These results show that neutrophil activation occurring during inflammation and sepsis could be detected by plasma MPO concentration. CONCLUSION: The plasma MPO concentrations may be a marker of the neutrophil proliferation and severity of inflammation.

Anti-platelet effects of Curcuma oil in experimental models of myocardial ischemia-reperfusion and thrombosis.

Extensive research on the mechanism of action and medicinal importance of curcumin obtained from turmeric (Curcuma longa) has unfolded its potential therapeutic value against many chronic ailments. Curcuma oil (C.oil), the highly lipophilic component from Curcuma longa has been documented for its neuroprotective efficacy against rat cerebral ischemia-reperfusion injury; however its effect on myocardial reperfusion injury remains unexplored. In the present study, effect of C.oil (500 mg/kg, po) was evaluated against myocardial ischemia-reperfusion induced injury in the rat model. C.oil failed to confer protection against cardiac injury, however significant reversal of ADP induced platelet aggregation (p<0.05) was evident in the same animals. Moreover, collagen and thrombin induced platelet aggregation (p<0.001) as well as tyrosine phosphorylation of various proteins in activated platelets was also suppressed. C.oil also offered significant protection against collagen-epinephrine induced thromboembolism in mice as well as augmented total time to occlusion against FeCl(3) induced arterial thrombosis in rats. C.oil however had no effect on coagulation parameters (TT, PT and aPTT) and exerted a mild effect on the bleeding time. Bioavailability of C.oil, as assessed by monitoring ar-turmerone, α,ß-turmerone and curlone, was 13%, 11% and 7% respectively, indicating high systemic exposure. Moreover, longer mean residence time (MRT) of ar-turmerone (13.2h), α,ß-turmerone (11.6h) and Curlone (14.0 h) and plasma elimination half lives in the range of 5.5 to 7.2h correlated with single 500 mg/kg dose regimen of C.oil. In the present study, C.oil thus seems to be an efficacious and safe anti-platelet agent which was protective against intravascular thrombosis.

Ramipril-like activity of Spondias mombin linn against no-flow ischemia and isoproterenol-induced cardiotoxicity in rat heart.

The cardioprotective property of Spondias mombin (SM) was investigated and compared with that of the ACE inhibitor, ramipril. Alterations to markers of myocardial injury and indices of antioxidant capacity by isoproterenol (ISP) intoxication were significantly corrected in groups treated with SM. The inflammatory index was increased by 24% in ISP-intoxicated group compared with control (P < 0.001) but reduced in the groups administered ISP and treated with 100 or 250 mg/kg SM by 17% (P < 0.001) and 11% (P < 0.05) respectively. Serum lactate dehydrogenase activity and cholesterol level which were significantly increased in ISP-intoxicated group compared with control were reduced in groups administered ISP and treated with SM. Serum phosphate levels in groups administered ISP and treated with SM were significantly lower than values obtained for the ISP-intoxicated group (P < 0.001). Tissue catalase and superoxide dismutase activities as well as glutathione level were significantly increased in groups administered ISP and treated with SM compared to ISP-intoxicated group while MDA and nitrite levels were decreased. Disruption in the structure of cardiac myofibrils by ISP intoxication was reduced by treatment with SM. Comparable results were obtained for ramipril. These results are indicative of the potent cardioprotective property of SM.

Glycogen synthase kinase-3beta induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3.

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3beta has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3beta during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3beta, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3beta-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3beta activation, indicating that induction of GSK-3beta lies upstream of MLK3. In GSK-3beta-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3beta. The activation of MLK3 by GSK-3beta occurred via phosphorylation of MLK3 on two amino acid residues, Ser(789) and Ser(793), that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3beta was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3beta. Taken together, our data provide a direct link between GSK-3beta and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3beta. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.

Negative regulation of mixed lineage kinase 3 by protein kinase B/AKT leads to cell survival.

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates c-jun N-terminal kinase (JNK) and can induce cell death in neurons. By contrast, the activation of phosphatidylinositol 3-kinase and AKT/protein kinase B (PKB) acts to suppress neuronal apoptosis. Here, we report a functional interaction between MLK3 and AKT1/PKBalpha. Endogenous MLK3 and AKT1 interact in HepG2 cells, and this interaction is regulated by insulin. The interaction domain maps to the C-terminal half of MLK3 (amino acids 511-847), and this region also contains a putative AKT phosphorylation consensus sequence. Endogenous JNK, MKK7, and MLK3 kinase activities in HepG2 cells are significantly attenuated by insulin treatment, whereas the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin reversed the effect. Finally, MLK3-mediated JNK activation is inhibited by AKT1. AKT phosphorylates MLK3 on serine 674 both in vitro and in vivo. Furthermore, the expression of activated AKT1 inhibits MLK3-mediated cell death in a manner dependent on serine 674 phosphorylation. Thus, these data provide the first direct link between MLK3-mediated cell death and its regulation by a cell survival signaling protein, AKT1.

Activation of the Drosophila MLK by ceramide reveals TNF-alpha and ceramide as agonists of mammalian MLK3.

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity remain unknown. Here, we demonstrate ceramide as the activator of Drosophila MLK (dMLK) and identify ceramide and TNF-alpha as agonists of mammalian MLK3. dMLK and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of dMLK and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-alpha also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of dMLK and MLK3, and MLK3 contributes to JNK activation induced by TNF-alpha.