![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CARDIOVASCULAR
Department of Pharmacology, School of Medicine, University of Toyama, Toyama, Japan (N.M., Y.T., Y.Hat.); and Department of Anesthesiology, Osaka University Faculty of Medicine, Osaka, Japan (Y.Hay.)
Received June 22, 2006; accepted September 25, 2006.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
), a potential role of eNOS in the pathophysiology of sepsis is unsubstantiated. Previous work using eNOS knockout mice has shown that they respond to endotoxin in an identical manner to wild-type littermates (Shesely et al., 1996), and it has been recently shown that less abundant expression of iNOS in LPS-treated eNOS knockout mice suggests that eNOS-derived NO is vital for facilitating iNOS expression in sepsis (Connelly et al., 2005). However, it has been revealed that mice overexpressing eNOS generate similar levels of plasma nitrite and nitrate to control animals in response to LPS but display resistance to LPS-induced hypotension, lung injury, and death (Yamashita et al., 2000). Moreover, we have recently demonstrated that eNOS expression is obviously diminished in blood vessels from rabbits (Matsuda et al., 2003) and in lung tissues from mice (Matsuda et al., 2004) following induction of sepsis with LPS. Because the relatively small amount of NO generated by eNOS provides important physiologic regulations, which are suggested to include those of vascular tone, bronchial tone, neurotransmission, or immune defense (Moncada et al., 1991; Gaston et al., 1994), impairment of expression and activity of eNOS, if any, may contribute to tissue injury in sepsis by hindrance of physiologic regulatory events with the involvement of eNOS-derived NO.
Activation of PI3-K is an important proximal event in LPS signaling. Cell stimulation with LPS activates PI3-K and its downstream effector, Akt (Monick et al., 2001; Guha and Mackman, 2002; Connelly et al., 2005). Akt has been shown to phosphorylate eNOS, leading to a persistent, Ca2+-independent enzyme activation (Dimmeler et al., 1999; Fulton et al., 1999). In accordance with this, Akt may play a key role in promoting endothelial NO synthesis. However, little is known about Akt-dependent eNOS phosphorylation in vascular endothelium following sepsis induction.
HMG-CoA reductase inhibitors are widely used for treatment of hyperlipidemia. This group of drugs is familiarly known as "statin," and the drugs' common mechanism of action is their ability to block hepatic conversion of HMG-CoA to L-mevalonate in the cholesterol biosynthetic pathway (Goldstein and Brown, 1990). However, a growing body of data suggest that HMG-CoA reductase inhibitors exhibit important immunomodulatory effects that are independent of their lipid-lowering ability (Kwak et al., 2000). These pleiotropic effects include anti-inflammatory actions, improvement of endothelial and microvascular functions, and modulation of eNOS (Lefer, 2002; Walter et al., 2004). Indeed, HMG-CoA reductase inhibitors have been shown to reverse the down-regulation of eNOS expression by hypoxia and oxidized low-density lipoprotein under cholesterol-clamped conditions (Laufs et al., 1997, 1998). Interestingly, treatment with an HMG-CoA reductase inhibitor, such as simvastatin, can profoundly improve survival in a murine model of sepsis (Merx et al., 2004). In the light of our previous report showing the diminished expression levels of eNOS in blood vessels from the animals after sepsis induction with LPS (Matsuda et al., 2003), it may be inferable that HMG-CoA reductase inhibitors could exert a protective effect against sepsis, at least in part, by virtue of their ability to ameliorate eNOS expression profile of endothelial cells, thereby preventing vascular events.
The present study was aimed at verifying vascular changes in the signaling cascade mechanisms for Akt-dependent phosphorylation of eNOS in an in vivo rabbit model of endotoxin challenge. In addition, we determined the therapeutic effect of the HMG-CoA reductase inhibitor fluvastatin on eNOS expression, eNOS phosphorylation, and activation of the PI3-K/Akt pathway in vascular tissues of this septic model.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
, 2002). All animals received care in compliance with the approved institutional animal care guidelines.
Drug Administration. Rabbits received three intraperitoneal injections of 5 mg/kg fluvastatin at 8-h intervals from 16 h before LPS treatment. This dose of fluvastatin was chosen because fluvastatin at this dose reportedly ameliorates endothelial dysfunction in obese Zucker rats (Nishimatsu et al., 2005). Fluvastatin was a gift from Novartis Pharma AG (Basel, Switzerland). It was dissolved in ethanol at a concentration of 20 mg/ml and diluted with saline at a rate of 1:3 to yield a final concentration of 5 mg of fluvastatin/ml of carrier. A control group of animals received the carrier only (carrier control). When wortmannin (1 mg/kg; BIOMOL International, Plymouth Meeting, PA) was used, it was intravenously given to the animal 1 h after LPS.
Preparation of Tissue Protein Extracts. At indicated times after injection of LPS or vehicle, the main and first branches of the mesenteric artery were carefully removed under induction of terminal anesthesia with pentobarbital, and the vessels were immediately frozen with liquid nitrogen. The blood vessels were then placed in ice-cold sterile water containing phosphatase and protease inhibitor cocktail (0.2 µl/100 mg of tissue; Sigma, St. Louis, MO), with the exception of the experiments using anti-mouse protein phosphatase 2A, and cleaned of adhering fat and connective tissue under a microscope. Thereafter, the tissues were powdered under liquid nitrogen and solubilized in 3 ml of ice-cold sterile water that contained 0.1% Triton X-100. The lysates were centrifuged at 1000gmax for 10 min at 4°C to pellet any insoluble material. The membrane fractions were prepared as described previously (Matsuda et al., 1999). Thus, the supernatant was then spun at 100,000gmax for 30 min at 4°C. The membrane pellet was resuspended in 100 µl of lysis buffer and saved. Protein concentrations were measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).
Primary Antibodies Used. Immunoblotting was performed with the following commercially available antibodies: anti-goat eNOS (R&D Systems, Minneapolis, MN), anti-mouse phospho-eNOS (Ser 1177) (BIOMOL International), anti-mouse Akt (BIOMOL International), anti-rabbit phospho-Akt 1/2/3 (Thr308) (Acris Antibodies GmbH, Hiddenhausen, Germany), anti-mouse phosphor-Akt 1 (Ser 473) (Acris Antibodies GmbH), anti-mouse protein phosphatase 2A (Upstate Cell Signaling, Lake Placid, NY), anti-mouse PI3-K p85
(GeneTex, San Antonio, TX), anti-mouse PI3-K p110 (BD Biosciences, San Jose, CA), anti-mouse PI3-K p85
(Acris Antibodies GmbH), and anti-mouse actin (GeneTex).
Western Blot Analysis. Samples (2–20 µg) were run on SDS-polyacrylamide gel electrophoresis, using 7.5 to 12.5% polyacrylamide gel, and electrotransferred to polyvinylidene difluoride filter membrane. To reduce nonspecific binding, the membrane was blocked for 60 min at room temperature in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4) containing 1% bovine serum albumin. The membrane was then incubated for 60 min at 4°C with primary antibodies diluted at 1:100 to 1:500 in PBS containing 0.05% Tween 20. After an extensive washing with PBS-Tween buffer, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse (eBioscience, San Diego, CA), anti-goat (eBioscience), or anti-rabbit (Bio-Rad Laboratories), antibody diluted at 1:1000 to 1:6000 in PBS-Tween buffer at room temperature for 60 min. The blots were washed twice in PBS-Tween buffer, developed using the enhanced chemiluminescence detection system (Amersham, Little Chalfont, Buckinghamshire, UK), exposed to X-ray film, and analyzed by NIH Image software produced by Wayne Rasband (National Institutes of Health, Bethesda, MD). To check for protein loading/transfer variations, all blots were stained with Ponceau red (washable, before incubation with antibodies) and with Coomassie Brilliant Blue (permanent, after the enhanced chemiluminescence detection system). Intensity of total protein bands per lane was evaluated by densitometry. Negligible loading/transfer variation was observed between samples.
Survival Studies in Sepsis. Additional groups of rabbits underwent LPS injection and were included in survival studies. Rabbits were randomly divided into two groups. One group of the animals that underwent LPS injection was treated with fluvastatin in the same way as described above. The animals were allowed free access to food and water, and the survival time of each rabbit was recorded for 60 h.
Statistical Analysis. All data were presented in terms of means ± S.E. Statistical assessment of the data were made by Student's t test or one-way analysis of variance followed by Bonferroni's multiple comparison test when appropriate. A P value <0.05 was considered statistically significant for all analyses.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
), a time-dependent decrease in the eNOS protein level was observed in mesenteric artery from the rabbit when sepsis was induced by intravenous injection of 100 µg/kg LPS (Fig. 1). Furthermore, sepsis induction dramatically reduced phosphorylation of native eNOS at Ser1177, as determined by Western blotting with the phosphospecific antibody. The ratio of phospho-eNOS to total eNOS decreased by 36 to 68% after 6 h and by 70 to 86% after 10 h.
|
; Fulton et al., 1999), the downstream phosphorylation of Akt was also studied under the same condition. The results of Western blot analysis revealed that vascular expression of Akt, dually phosphorylated at Thr308 and Ser473, was reduced in a time-dependent manner after induction of sepsis by LPS (Fig. 2A). Immunoblot analysis with antibodies against total Akt showed equal protein levels in all samples, thus demonstrating that the reduced amount of phosphorylated Akt following sepsis induction was not due to decreased expression of Akt. Indeed, a marked time-dependent reduction in the ratio of phospho-Akt to total Akt was found after sepsis induction (Fig. 2, B and C).
|
). The expression level of protein phosphatase 2A was examined in vascular tissues from septic rabbits. Immunoblot analysis showed a slight but significant decrease rather than an increase in vascular expression of protein phosphatase 2A with the progression of sepsis (83 ± 4% of control at 10 h after LPS, n = 5, P < 0.05).
Akt is located downstream of PI3-K (Datta et al., 1996; Klippel et al., 1997). Although the PI3-K family comprises at least 12 members (Vanhaesebroeck and Waterfield, 1999), we assessed PI3-K protein levels by Western blotting using its specific antibodies because of the importance of the form in the regulation of downstream signaling molecules (Bi et al., 1999). As documented, PI3-K is a heterodimer phospholipid kinase composed of an 85-kDa regulatory subunit and 110-kDa catalytic subunit (Carpenter et al., 1990); the 85- and 110-kDa bands that corresponded to the PI3-K heterodimer were detected in rabbit mesenteric artery (Fig. 3). The total amount of the PI3-K heterodimer was substantially unchanged by sepsis induction. The protein kinase activity of PI3-K was determined by assessment of translocation of PI3-K to the membrane pool. Sepsis induction resulted in a time-dependent reduction in the amount of PI3-K heterodimer in the membrane fraction in vascular tissues (Fig. 3), suggesting that PI3-K was much less activated with the development of sepsis.
|
protein levels during sepsis. Western blot analysis using the specific antibody against the 85-kDa subunit of PI3-K was performed in rabbit mesenteric artery. Similar to PI3-K, the PI3-K 85-kDa subunit in the membrane/total fractions was reduced by 34% after 6 h and by 20% after 10 h of sepsis (n = 3).
Statins may increase eNOS phosphorylation and expression through the PI3-K/Akt pathway (Kureishi et al., 2000). Effects of in vivo treatment of rabbits with fluvastatin on vascular expression and phosphorylation of Akt and eNOS were tested under control and septic conditions. The total expression levels of Akt and eNOS in vascular tissues were unchanged by in vivo fluvastatin treatment of control rabbits. However, fluvastatin induced a 2.6- and 2-fold increase in phospho-Akt and phosphor-eNOS levels in vascular tissues from control rabbits, respectively (Fig. 4). Moreover, fluvastatin strongly prevented the prominent reduction in Akt and eNOS phosphorylation caused by 10 h of LPS-induced sepsis (8.9- and 9.6-fold increase, respectively) (Fig. 4). In addition, fluvastatin significantly improved a sepsis-induced reduction in total eNOS expression from 54 ± 3% to 85 ± 3% of control.
|
The reduced amount of PI3-K heterodimer in the membrane fraction in vascular tissues 10 h after induction of sepsis was significantly but incompletely attenuated by in vivo treatment with fluvastatin (Fig. 5). When the PI3-K inhibitor wortmannin (1 mg/kg) was given to the rabbit 1 h after LPS, this treatment resulted in a partial inhibition of the fluvastatin-induced increase in phosphorylation of Akt (Fig. 6). Furthermore, wortmannin treatment partially inhibited the fluvastatin-induced increase in phosphorylation of eNOS in septic vascular tissues (Fig. 7). The increased protein expression level of total eNOS in septic vascular tissues under fluvastatin therapy was significantly lowered by wortmannin, but this lowering effect was small (Fig. 7).
|
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). In addition, the data presented in this study demonstrated that sepsis caused a progressive and profound reduction in phosphorylation of eNOS in rabbit mesenteric artery. Because phosphorylation of eNOS results in increased NO production, our results would suggest less production of NO by eNOS in sepsis. NO produced by eNOS is considered to serve as a ubiquitous mediator molecule involved in many physiologic regulations, including vascular tone, neurotransmission, and immune defense (Moncada et al., 1991; Gaston et al., 1994). Therefore, loss of eNOS-based NO synthesis may well contribute to vascular abnormalities, inflammatory sequelae, and physiologic dysfunction of organs in sepsis. Indeed, particular attention has been focused on endothelial cells, which are a major target of sepsis-induced events and whose dysfunction plays a crucial role in the pathophysiology of septic shock (Peters et al., 2003).
The PI3-K/Akt pathway has been described to signal eNOS activation in response to shear stress (Dimmeler et al., 1999; Fulton et al., 1999). Thus, eNOS phosphorylation is downstream to Akt activation. Our study suggests that the sepsis-induced decrease in eNOS phosphorylation may be the result of reduced phosphorylation of Akt, because induction of sepsis was found to lead to a time dependently marked reduction in Akt phosphorylation in rabbit mesenteric artery without any change in the total amount of Akt. A role of protein phosphatase 2A in dephosphorylation of Akt has been documented in vascular smooth muscle cells in response to the cellular stress condition (Yellaturu et al., 2002). This does not seem to be the case, however, as vascular expression of protein phosphatase 2A was decreased rather than increased following sepsis induction; we cannot completely exclude altered activity of protein phosphatase 2A that could be regulated not only by its expression but through other mechanisms. Activation of Akt occurs downstream of PI3-K (Datta et al., 1996; Klippel et al., 1997). Thus, activated PI3-K catalyzes phosphorylation of membrane inositol lipids and causes accumulation of phosphatidylinositol 3,4,5-trisphosphate and its phospholipid phosphatase product phosphatidylinositol 3,4-bisphosphate in the membrane. Such membrane changes allow docking of the lipid kinases phosphatidylinositol-dependent kinase 1 and Akt. Although PI3-K is a heterodimer lipid kinase consisting of an 85-kDa subunit bound to a 110-kDa catalytic subunit (Carpenter et al., 1990), the amounts of both subunits in the membranous pool were progressively and profoundly decreased in rabbit mesenteric artery following induction of sepsis. The mechanism of the decrease is probably due to a decrease in translocation. This possibility could be supported by the lack of changes in the total amount of the PI3-K heterodimer during sepsis, although it should be kept in mind that other subunits of class IA PI3-K family members (Vanhaesebroeck and Waterfield, 1999) may be involved in sepsis-induced changes in this pathway. It is essential for PI3-K activation to allow translocation of the cytosolic PI3-K to the membranes where its lipid substrates and its binding monomeric G protein Ras reside (Vanhaesebroeck and Waterfield, 1999). Akt is activated following PI3-K recruitment to the inner surface of the plasma membrane. Therefore, it would be reasonable from the present data to conclude that PI3-K was much less activated with the development of sepsis. We thus indicate that impaired phosphorylation of Akt in vascular tissues during sepsis seems to be attributed to reduced activity of its upstream enzyme PI3-K.
Contrary to the present findings in mesenteric arteries from septic rabbits, in vitro studies have shown that LPS stimulation caused phosphorylation of Akt in human alveolar macrophages (Monick et al., 2001), human monocytic cells (Guha and Mackman, 2002), and human umbilical vein endothelial cells (Connelly et al., 2005), although the time dependence of LPS-induced Akt phosphorylation for each is quite different. At the present time, we do not have a clear understanding to reconcile these findings. However, the PI3-K/Akt pathway has been shown to regulate negatively nuclear factor-
B, a transcription factor activated by LPS, and overexpression of proinflammatory genes (Romashkova and Makarov, 1999; Vanhaesebroeck and Waterfield, 1999). Inhibition of PI3-K by wortmannin enhances LPS-induced NOS in murine peritoneal macrophages (Park et al., 1997), and activation of PI3-K/Akt can suppress LPS-induced lipoprotein lipase expression in J774.2 macrophages (Tengku-Muhammad et al., 1999). Induction of NOS by LPS in C6 glial cells and rat primary astrocytes has also been found to be regulated negatively by activation of PI3-K (Pahan et al., 1999). Finally, overexpression of Akt has been documented to improve survival in cecal ligation and puncture septic mouse model in association with prevention of sepsis-induced lymphocyte apoptosis (Bommhardt et al., 2004).
Interestingly, the HMG-CoA reductase inhibitor fluvastatin restored eNOS phosphorylation, which was markedly impaired in mesenteric artery from septic rabbits, and this improvement was associated with the recovery of Akt phosphorylation. It is well established that Akt-dependent phosphorylation of eNOS is necessary for a full activation of eNOS and endothelium-dependent vasorelaxation (Dimmeler et al., 1999; Fulton et al., 1999). Statins can induce angiogenesis via activation of Akt and eNOS in vascular endothelial cells (Kureishi et al., 2000). Furthermore, statins seem to stimulate membrane translocation of Akt and its activation in endothelial cells (Skaletz-Rorowski et al., 2003). Thus, our results suggest that sepsis-induced impairment of eNOS activation showed a substantial improvement by in vivo treatment with fluvastatin, at least in part, via its direct effect on Akt. However, we found that fluvastatin treatment was capable of attenuating the reduction in membrane translocations of the PI3-K heterodimer in septic vascular tissues, although this effect was not so pronounced. Moreover, in vivo treatment with the PI3-K inhibitor wortmannin led to a partial inhibition of increased phosphorylation of Akt and eNOS by fluvastatin treatment of the septic rabbit. These results suggest that the stimulatory effect of fluvastatin on Akt phosphorylation may partially involve PI3-K activation. Indeed, it has previously been demonstrated that inhibition of the PI3-K pathway by wortmannin and the additional PI3-K inhibitor LY294002 inhibited endothelial progenitor cell differentiation in mice treated with statins (Dimmeler et al., 2001). Furthermore, LY294002 could reportedly attenuate the increase in the level of heme oxygenase-1 by simvastatin in human and rat aortic smooth muscle cells (Lee et al., 2004).
We also found that fluvastatin restored vascular eNOS expression, which was diminished following sepsis induction. Wortmannin treatment resulted in a significant inhibition of this restoration, suggesting that the effect of fluvastatin on total eNOS protein expression in septic vascular tissues may involve improvement of the PI3-K/Akt pathway. However, the inhibitory effect of wortmannin on the fluvastatin-induced increase in total eNOS in septic vascular tissues was evidently small. Statins inhibit the Rho-dependent pathway via suppression of Rho geranylgeranylation, which is an important post-translational modification and is required for the membrane translocation and activation of Rho, and consequently stimulate eNOS expression (Laufs et al., 1998). It is conceivable that the recovery effect of fluvastatin on eNOS expression may be due to the inhibition of the Rho-dependent pathway; however, further investigations are needed to prove this point. It would thus be expected that fluvastatin can improve sepsis-induced deteriorations of eNOS expression and activation, thereby ameliorating vascular endothelial dysfunction in sepsis. The ability of statins to restore the pathological endothelial dysfunction has been documented in animal models of stroke (Laufs et al., 2000) and diabetes (Nishimatsu et al., 2005).
Our survival studies clearly showed that fluvastatin protected the rabbits against LPS-induced septic death. This finding is in good agreement with the result by Merx et al. (2004), who have reported that simvastatin significantly improves survival in a cecal ligation and puncture septic mouse model. They have stated the importance of increased mononuclear cell adhesiveness in septic mice to be reversed by simvastatin treatment (Merx et al., 2004). In light of the recent report that overexpression of Akt can improve survival in sepsis (Bommhardt et al., 2004), we interpret the present results to suggest that the profound improvement in survival may be associated with up-regulation of the Akt pathway by statins. However, other mechanisms, which may also involve important cellular effects independent of lipid lowering, cannot be excluded.
In summary, phosphorylation of eNOS was markedly diminished in rabbit mesenteric artery following LPS-induced sepsis. This seemed to be associated with the impaired PI3-K/Akt pathway, although direct experimental evidence to support this mechanism awaits further study. Fluvastatin restored sepsis-induced impairment of eNOS phosphorylation in association with the recovery of Akt phosphorylation and dramatically improved survival in sepsis. Although being well established in the treatment of lipid disorders, statins may have a new potential of being therapeutic for subjects with sepsis.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: iNOS, inducible nitric-oxide synthase; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; LPS, lipopolysaccharide; PI3-K, phosphatidylinositol 3-kinase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; PBS, phosphate-buffered saline LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one; Flv, fluvastatin.
Address correspondence to: Dr. Yuichi Hattori, Department of Pharmacology, School of Medicine, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan. E-mail: yhattori{at}med.u-toyama.ac.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bi L, Okabe I, Bernard DJ, Wynshaw-Boris A, and Nussbaum RL (1999) Proliferative defect and embryonic lethality in mice homozygous for a deletion in the p110 subunit of phosphoinositide 3-kinase. J Biol Chem 274: 10963–10968.
Bommhardt U, Chang KC, Swanson PE, Wagner TH, Tinsley KW, Karl IE, and Hotchkiss RS (2004) Akt decreases lymphocyte apoptosis and improves survival in sepsis. J Immunol 172: 7583–7591.
Carpenter CL, Duckworth BC, Auger KR, Cohen B, Schaffhausen BS, and Cantley LC (1990) Purification and characterization of phosphoinositide 3-kinase from rat liver. J Biol Chem 265: 19704–19711.
Connelly L, Madhani M, and Hobbs AJ (2005) Resistance to endotoxic shock in endothelial nitric-oxide synthase (eNOS) knock-out mice. A pro-inflammatory role for eNOS-derived NO in vivo. J Biol Chem 280: 10040–10046.
Datta K, Bellacosa A, Chan TO, and Tsichlis PN (1996) Akt is a direct target of the phosphatidylinositol 3-kinase: activation by growth factors, v-src and v-Ha-ras, in Sf9 and mammalian cells. J Biol Chem 271: 30835–30839.
Dimmeler S, Aicher A, Vasa M, Mildner-Rihm C, Adler K, Tiemann M, Rütten H, Fichtlscherer S, Martin H, and Zeiher AM (2001) HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI3-kinase/Akt pathway. J Clin Investig 108: 391–397.[CrossRef][Medline]
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, and Zeiher AM (1999) Activation of nitric oxide synthase in endothelial cells via Akt-dependent phosphorylation. Nature (Lond) 399: 601–605.[CrossRef][Medline]
Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, and Sessa WC (1999) Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature (Lond) 399: 597–601.[CrossRef][Medline]
Gaston B, Drazen JM, Loscalzo J, and Stamler JS (1994) The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 149: 538–551.[Abstract]
Goldstein JL and Brown MS (1990) Regulation of the mevalonate pathway. Nature (Lond) 343: 425–430.[CrossRef][Medline]
Guha M and Mackman N (2002) The phosphatidylinositol 3-kinase-Akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells. J Biol Chem 277: 32124–32132.
Klippel A, Kavanaugh WM, Pot D, and Williams LT (1997) A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin domain. Mol Cell Biol 17: 338–344.[Abstract]
Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, and Walsh K (2000) The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med 6: 1004–1010.[CrossRef][Medline]
Kwak B, Mulhaupt F, Myit S, and Mach F (2000) Statins as a newly recognized type of immunomodulator. Nat Med 6: 1399–1402.[CrossRef][Medline]
Laufs U, Gertz K, Huang P, Nickenig G, Böhm M, Dirnagl U, and Endres M (2000) Atorvastatin upregulates type III nitric oxide synthase in thrombocytes, decreases platelet activation, and protects from cerebral ischemia in normocholesterolemic mice. Stroke 31: 2437–2449.
Laufs U, La Fata V, and Liao JK (1997) Inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase blocks hypoxia-mediated down-regulation of endothelial nitric oxide synthase. J Biol Chem 272: 31725–31729.
Laufs U, La Fata V, Plutzky J, and Liao JK (1998) Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97: 1129–1135.
Lee T-S, Chang C-C, Zhu Y, and Shyy Y-J (2004) Simvastatin induced heme oxygenase-1. A novel mechanism of vessel protection. Circulation 110: 1296–1302.
Lefer DJ (2002) Statins as potent antiinflammatory drugs. Circulation 106: 2041–2042.
Matsuda N, Hattori Y, Akaishi Y, Suzuki Y, Kemmotsu O, and Gando S (2000) Impairment of cardiac -adrenoceptor cellular signaling by decreased expression of Gs in septic rabbits. Anesthesiology 93: 1465–1473.[CrossRef][Medline]
Matsuda N, Hattori Y, Gando S, Akaishi Y, Kemmotsu O, and Kanno M (1999) Diabetes-induced down-regulation of 1-adrenoceptor mRNA expression in rat heart. Biochem Pharmacol 58: 881–885.[CrossRef][Medline]
Matsuda N, Hattori Y, Sakuraya F, Kobayashi M, Zhang X-H, Kemmotsu O, and Gando S (2002) Hemodynamic significance of histamine synthesis and histamine H1- and H2-receptor gene expression during endotoxemia. Naunyn-Schmiedeberg's Arch Pharmacol 366: 513–521.[CrossRef][Medline]
Matsuda N, Hattori Y, Takahashi Y, Nishihira J, Jesmin S, Kobayashi M, and Gando S (2004) Therapeutic effect of in vivo transfection of transcription factor decoy to NF-B on septic lung in mice. Am J Physiol 287: L1248–L1255.
Matsuda N, Hattori Y, Zhang X-H, Fukui H, Kemmotsu O, and Gando S (2003) Contractions to histamine in pulmonary and mesenteric arteries from endotoxemic rabbits: modulation by vascular expressions of inducible nitric-oxide synthase and histamine H1-receptors. J Pharmacol Exp Ther 307: 175–181.
Merx MW, Liehn EA, Janssens U, Lütticken R, Schrader J, Hanrath P, and Weber C (2004) HMG-CoA reductase inhibitor simvastatin profoundly improves survival in a murine model of sepsis. Circulation 109: 2560–2565.
Moncada S, Palmer PMJ, and Higgs EA (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109–142.[Medline]
Monick MM, Carter AB, Robeff PK, Flaherty DM, Peterson MW, and Hunninghake GW (2001) Lipopolysaccharide activates Akt in human alveolar macrophages resulting in nuclear accumulation and transcriptional activity of -catenin. J Immunol 166: 4713–4720.
Nishimatsu H, Suzuki E, Satonaka H, Takeda R, Omata M, Fujita T, Nagai R, Kitamura T, and Hirata Y (2005) Endothelial dysfunction and hypercontractility of vascular myocytes are ameliorated by fluvastatin in obese Zucker rats. Am J Physiol 288: 1770–1776.
Pahan K, Raymond JR, and Singh I (1999) Inhibition of phosphatidylinositol 3-kinase induces nitric-oxide synthase in lipopolysaccharide- or cytokine-stimulated C6 glial cells. J Biol Chem 274: 7528–7536.
Park YC, Lee CH, Kang HS, Chung HT, and Kim HD (1997) Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, enhances LPS-induced NO production from murine peritoneal macrophages. Biochem Biophys Res Commun 240: 692–696.[CrossRef][Medline]
Peters K, Unger RE, Brunner J, and Kirkpatrick CJ (2003) Molecular basis of endothelial dysfunction in sepsis. Cardiovasc Res 60: 49–57.
Romashkova JA and Makarov SS (1999) NF-kB is a target of AKT in anti-apoptotic PDGF signaling. Nature (Lond) 401: 86–90.[CrossRef][Medline]
Shesely EG, Maeda N, Kim HS, Desai KM, Krege JH, Laubach VE, Sherman PA, Sessa WC, and Smithies O (1996) Elevated blood pressure in mice lacking endothelial nitric oxide synthase. Proc Natl Acad Sci USA 93: 13176–13181.
Skaletz-Rorowski A, Lutchman M, Kureishi Y, Lefer DJ, Faust JR, and Walsh K (2003) HMG-CoA reductase inhibitors promote cholesterol-dependent Akt/PKB translocation to membrane domains in endothelial cells. Cardiovasc Res 57: 253–264.
Tengku-Muhammad TS, Hughes TR, Cryer A, and Ramji DP (1999) Involvement of both the tyrosine kinase and the phosphatidylinositol-3' kinase signal transduction pathways in the regulation of lipoprotein lipase expression in J774.2 macrophages by cytokines and lipopolysaccharide. Cytokine 11: 463–468.[CrossRef][Medline]
Titheradge MA (1999) Nitric oxide in septic shock. Biochim Biophys Acta 1411: 437–455.[Medline]
Vanhaesebroeck B and Waterfield MD (1999) Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253: 239–254.[CrossRef][Medline]
Walter DH, Dimmeler S, and Zeiher AM (2004) Effects of statins on endothelium and endothelial progenitor cell recruitment. Semin Vasc Med 4: 385–393.[CrossRef][Medline]
Yamashita T, Kawashima S, Ohashi Y, Ozaki M, Ueyama T, Ishida T, Inoue N, Hirata K, Akita H, and Yokoyama M (2000) Resistance to endotoxin shock in transgenic mice overexpressing endothelial nitric oxide synthase. Circulation 101: 931–937.
Yellaturu CR, Bhanoori M, Neeli I, and Rao GN (2002) N-Ethylmaleimide inhibits platelet-derived growth factor BB-stimulated Akt phosphorylation via activation of protein phosphatase 2A. J Biol Chem 277: 40148–40155.
This article has been cited by other articles:
|
|
 |
|
  H. Kinoshita, N. Matsuda, H. Kaba, N. Hatakeyama, T. Azma, K. Nakahata, Y. Kuroda, K. Tange, H. Iranami, and Y. Hatano Roles of Phosphatidylinositol 3-Kinase-Akt and NADPH Oxidase in Adenosine 5'-Triphosphate-Sensitive K+ Channel Function Impaired by High Glucose in the Human Artery Hypertension, September 1, 2008; 52(3): 507 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Matsuda, Y. Takano, S.-i. Kageyama, N. Hatakeyama, K. Shakunaga, I. Kitajima, M. Yamazaki, and Y. Hattori Silencing of caspase-8 and caspase-3 by RNA interference prevents vascular endothelial cell injury in mice with endotoxic shock Cardiovasc Res, October 1, 2007; 76(1): 132 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-J. Zhang, H. Wei, T. Hagen, and B. Frei {alpha}-Lipoic acid attenuates LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway PNAS, March 6, 2007; 104(10): 4077 - 4082. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. McGown and Z. L. S. Brookes Beneficial effects of statins on the microcirculation during sepsis: the role of nitric oxide Br. J. Anaesth., February 1, 2007; 98(2): 163 - 175. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
