Sustained Oxidative Stress Causes Late Acute Renal Failure via Duplex Regulation on p38 MAPK and Akt Phosphorylation in Severely Burned Rats

Background

Clinical evidence indicates that late acute renal failure (ARF) predicts high mortality in severely burned patients but the pathophysiology of late ARF remains undefined. This study was designed to test the hypothesis that sustained reactive oxygen species (ROS) induced late ARF in a severely burned rat model and to investigate the signaling mechanisms involved.

Materials and Methods

Rats were exposed to 100°C bath for 15 s to induce severe burn injury (40% of total body surface area). Renal function, ROS generation, tubular necrosis and apoptosis, and phosphorylation of MAPK and Akt were measured during 72 hours after burn.

Results

Renal function as assessed by serum creatinine and blood urea nitrogen deteriorated significantly at 3 h after burn, alleviated at 6 h but worsened at 48 h and 72 h, indicating a late ARF was induced. Apoptotic cells and cleavage caspase-3 in the kidney went up slowly and turned into significant at 48 h and 72 h. Tubular cell ROS production shot up at 6 h and continuously rose during the 72-h experiment. Scavenging ROS with tempol markedly attenuated tubular apoptosis and renal dysfunction at 72 h after burn. Interestingly, renal p38 MAPK phosphorylation elevated in a time dependent manner whereas Akt phosphorylation increased during the first 24 h but decreased at 48 h after burn. The p38 MAPK specific inhibitor SB203580 alleviated whereas Akt inhibitor exacerbated burn-induced tubular apoptosis and renal dysfunction. Furthermore, tempol treatment exerted a duplex regulation through inhibiting p38 MAPK phosphorylation but further increasing Akt phosphorylation at 72 h postburn.

Conclusions

These results demonstrate that sustained renal ROS overproduction induces continuous tubular cell apoptosis and thus a late ARF at 72 h after burn in severely burned rats, which may result from ROS-mediated activation of p38 MAPK but a late inhibition of Akt phosphorylation.     

Activation of CD40 with platelet derived CD154 promotes reactive oxygen species dependent death of human hepatocytes during hypoxia and reoxygenation

Background

Hypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis.

Methods

Human hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2′,7′-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay.

Results

Exposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis.

Conclusions

CD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.     

Lin28a Protects against Hypoxia/Reoxygenation Induced Cardiomyocytes Apoptosis by Alleviating Mitochondrial Dysfunction under High Glucose/High Fat Conditions

Aim

The aim of the present study was to investigate the role of Lin28a in protecting against hypoxia/reoxygenation (H/R)-induced cardiomyocytes apoptosis under high glucose/high fat (HG/HF) conditions.

Methods

Primary cardiomyocytes which were isolated from neonatal mouse were randomized to be treated with lentivirus carrying Lin28a siRNA, Lin28acDNA 72 h before H/R (9 h/2 h). Cardiomyocytes biomarkers release (LDH and CK), cardiomyocytes apoptosis, mitochondria biogenesis and morphology, intracellular reactive oxygen species (ROS) production, ATP content and inflammatory cytokines levels after H/R injury in high glucose/high fat conditions were compared between groups. The target proteins of Lin28a were examined by western blot analysis.

Results

Our results revealed that Lin28a cDNA transfection (overexpression) significantly inhibited cardiomyocyte apoptotic index, improved mitochondria biogenesis, increased ATP production and reduced ROS production as compared with the H/R group in HG/HF conditions. Lin28a siRNA transfection (knockdown) rendered the cardiomyocytes more susceptible to H/R injury as evidenced by increased apoptotic index, impaired mitochondrial biogenesis, decreased ATP production and increased ROS level. Interestingly, these effects of Lin28a were blocked by pretreatment with the PI3K inhibitor wortmannin. Lin28a overexpression increased, while Lin28a knockdown inhibited IGF1R, Nrf-1, Tfam, p-IRS-1, p-Akt, p-mTOR, p-p70s6k, p-AMPK expression levels after H/R injury in HG/HF conditions. Moreover, pretreatment with wortmannin abolished the effects of Lin28a on the expression levels of p-AKT, p-mTOR, p-p70s6k, p-AMPK.

Conclusions

The present results suggest that Lin28a inhibits cardiomyocytes apoptosis by enhancing mitochondrial biogenesis and function under high glucose/high fat conditions. The mechanism responsible for the effects of Lin28a is associated with the PI3K/Akt dependent pathway.     

Spry1 and Spry4 Differentially Regulate Human Aortic Smooth Muscle Cell Phenotype via Akt/FoxO/Myocardin Signaling

Background

Changes in the vascular smooth muscle cell (VSMC) contractile phenotype occur in pathological states such as restenosis and atherosclerosis. Multiple cytokines, signaling through receptor tyrosine kinases (RTK) and PI3K/Akt and MAPK/ERK pathways, regulate these phenotypic transitions. The Spry proteins are feedback modulators of RTK signaling, but their specific roles in VSMC have not been established.

Methodology/Principal Findings

Here, we report for the first time that Spry1, but not Spry4, is required for maintaining the differentiated state of human VSMC in vitro. While Spry1 is a known MAPK/ERK inhibitor in many cell types, we found that Spry1 has little effect on MAPK/ERK signaling but increases and maintains Akt activation in VSMC. Sustained Akt signaling is required for VSMC marker expression in vitro, while ERK signaling negatively modulates Akt activation and VSMC marker gene expression. Spry4, which antagonizes both MAPK/ERK and Akt signaling, suppresses VSMC differentiation marker gene expression. We show using siRNA knockdown and ChIP assays that FoxO3a, a downstream target of PI3K/Akt signaling, represses myocardin promoter activity, and that Spry1 increases, while Spry4 decreases myocardin mRNA levels.

Conclusions

Together, these data indicate that Spry1 and Spry4 have opposing roles in VSMC phenotypic modulation, and Spry1 maintains the VSMC differentiation phenotype in vitro in part through an Akt/FoxO/myocardin pathway.     

Lymphocyte apoptosis in murine Pneumocystis pneumonia

Background

Apoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due to Pneumocystis murina, an infection localized to the lungs.

Methods

Control mice and mice depleted of CD4+ lymphocytes were inoculated with Pneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins.

Results

In control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim.

Conclusion

Apoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response to Pneumocystis but is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins.     

Association Study to Evaluate FoxO1 and FoxO3 Gene in CHD in Han Chinese

Background

Coronary heart disease (CHD) is one of the leading causes of mortality and morbidity in China. Genetic factors that predispose individuals to CHD are unclear. In the present study, we aimed to determine whether the variation of FoxOs, a novel genetic factor associated with longevity, was associated with CHD in Han Chinese populations.

Methods

1271 CHD patients and 1287 age-and sex-matched controls from Beijing and Harbin were included. We selected four tagging single nucleotide polymorphisms (SNPs) of FoxO1 (rs2755209, rs2721072, rs4325427 and rs17592371) and two tagging SNPs of FoxO3 (rs768023 and rs1268165). And the genotypes of these SNPs were determined in both CHD patients and non-CHD controls.

Results

For population from Beijing, four SNPs of FoxO1 and two SNPs of FoxO3 were found not to be associated with CHD (p>0.05). And this was validated in the other population from Harbin (p>0.05). After combining the two geographically isolated case-control populations, the results showed that the six SNPs did not necessarily predispose to CHD in Han Chinese(p>0.05). In stratified analysis according to gender, the history of smoking, hypertension, diabetes mellitus, hyperlipidemia and the metabolic syndrome, we further explored that neither the variants of FoxO1 nor the variants of FoxO3 might be associated with CHD (p>0.05).

Conclusion

The variants of FoxO1 and FoxO3 may not increase the prevalence of CHD in Han Chinese population.     

Vinpocetine Attenuates Neointimal Hyperplasia in Diabetic Rat Carotid Arteries after Balloon Injury

Background

Diabetes exacerbates abnormal vascular smooth muscle cell (VSMC) accumulation in response to arterial wall injury. Vinpocetine has been shown to improve vascular remolding; however, little is known about the direct effects of vinpocetine on vascular complications mediated by diabetes. The objective of this study was to determine the effects of vinpocetine on hyperglycemia-facilitated neointimal hyperplasia and explore its possible mechanism.

Materials and Methods

Nondiabetic and diabetic rats were subjected to balloon injury of the carotid artery followed by 3-week treatment with either vinpocetine (10 mg/kg/day) or saline. Morphological analysis and proliferating cell nuclear antigen (PCNA) immunostaining were performed on day 21. Rat VSMCs proliferation was determined with 5-ethynyl-20-deoxyuridine cell proliferation assays. Chemokinesis was monitored with scratch assays, and production of reactive oxygen species (ROS) was assessed using a 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) flow cytometric assay. Apoptosis was detected by annexin V-FITC/PI flow cytometric assay. Cell signaling was assessed by immunblotting.

Results

Vinpocetine prevented intimal hyperplasia in carotid arteries in both normal (I/M ratio: 93.83 ± 26.45% versus 143.2 ± 38.18%, P<0.05) and diabetic animals (I/M ratio: 120.5 ± 42.55% versus 233.46 ± 33.98%, P<0.05) when compared to saline. The in vitro study demonstrated that vinpocetine significantly inhibited VSMCs proliferation and chemokinesis as well as ROS generation and apoptotic resistance, which was induced by high glucose (HG) treatment. Vinpocetine significantly abolished HG-induced phosphorylation of Akt and JNK1/2 without affecting their total levels. For downstream targets, HG-induced phosphorylation of IκBα was significantly inhibited by vinpocetine. Vinpocetine also attenuated HG-enhanced expression of PCNA, cyclin D1 and Bcl-2.

Conclusions

Vinpocetine attenuated neointimal formation in diabetic rats and inhibited HG-induced VSMCs proliferation, chemokinesis and apoptotic resistance by preventing ROS activation and affecting MAPK, PI3K/Akt, and NF-κB signaling.     

Akt regulates IL-10 mediated suppression of TNFα-induced cardiomyocyte apoptosis by upregulating Stat3 phosphorylation

Background

We have already reported that TNF-α increases cardiomyocyte apoptosis and IL-10 treatment prevented these effects of TNF-α. Present study investigates the role of Akt and Jak/Stat pathway in the IL-10 modulation of TNF-α induced cardiomyocyte apoptosis.

Methodology/Principal findings

Cardiomyocytes isolated from adult Sprague Dawley rats were exposed to TNF-α (10 ng/ml), IL-10 (10 ng/ml) and TNF-α+IL-10 (ratio 1) for 4 h. Exposure to TNF-α resulted in an increase in cardiomyocyte apoptosis as measured by flow cytometry and TUNEL assay. IL-10 by itself had no effect, but it prevented TNF-α induced apoptosis. IL-10 treatment increased Akt levels within cardiomyocytes and this change was associated with an increase in Jak1 and Stat3 phosphorylation. Pre-exposure of cells to Akt inhibitor prevented IL-10 induced Stat3 phosphorylation. Furthermore, in the presence of Akt or Stat3 inhibitor, IL-10 treatment was unable to block TNF-α induced cardiomyocyte apoptosis.

Conclusion

It is suggested that IL-10 modulation of TNF-α induced cardiomyocyte apoptosis is mediated by Akt via Stat3 activation.     

AMPK-Regulated and Akt-Dependent Enhancement of Glucose Uptake Is Essential in Ischemic Preconditioning-Alleviated Reperfusion Injury

Aims

Ischemic preconditioning (IPC) is a potent form of endogenous protection. However, IPC-induced cardioprotective effect is significantly blunted in insulin resistance-related diseases and the underlying mechanism is unclear. This study aimed to determine the role of glucose metabolism in IPC-reduced reperfusion injury.

Methods

Normal or streptozotocin (STZ)-treated diabetic rats subjected to 2 cycles of 5 min ischemia/5 min reperfusion prior to myocardial ischemia (30 min)/reperfusion (3 h). Myocardial glucose uptake was determined by ¹⁸F-fluorodeoxyglucose-positron emission tomography (PET) scan and gamma-counter biodistribution assay.

Results

IPC exerted significant cardioprotection and markedly improved myocardial glucose uptake 1 h after reperfusion (P<0.01) as evidenced by PET images and gamma-counter biodistribution assay in ischemia/reperfused rats. Meanwhile, myocardial translocation of glucose transporter 4 (GLUT4) to plasma membrane together with myocardial Akt and AMPK phosphorylation were significantly enhanced in preconditioned hearts. Intramyocardial injection of GLUT4 siRNA markedly decreased GLUT4 expression and blocked the cardioprotection of IPC as evidence by increased myocardial infarct size. Moreover, the PI3K inhibitor wortmannin significantly inhibited activation of Akt and AMPK, reduced GLUT4 translocation, glucose uptake and ultimately, depressed IPC-induced cardioprotection. Furthermore, IPC-afforded antiapoptotic effect was markedly blunted in STZ-treated diabetic rats. Exogenous insulin supplementation significantly improved glucose uptake via co-activation of myocardial AMPK and Akt and alleviated ischemia/reperfusion injury as evidenced by reduced myocardial apoptosis and infarction size in STZ-treated rats (P<0.05).

Conclusions

The present study firstly examined the role of myocardial glucose metabolism during reperfusion in IPC using direct genetic modulation in vivo. Augmented glucose uptake via co-activation of myocardial AMPK and Akt in reperfused myocardium is essential to IPC-alleviated reperfusion injury. This intrinsic metabolic modulation and cardioprotective capacity are present in STZ-treated hearts and can be triggered by insulin.     

Zerumbone suppresses IKKα, Akt,and FOXO1 activation,resulting in apoptosis of GBM 8401 cells

Background

Zerumbone, a sesquiterpene compound isolated from subtropical ginger, Zingiber zerumbet Smith, has been documented to exert antitumoral and anti- inflammatory activities. In this study, we demonstrate that zerumbone induces apoptosis in human glioblastoma multiforme (GBM8401) cells and investigate the apoptotic mechanism.

Methods

We added a caspase inhibitor and transfected wild-type (WT) IKK and Akt into GBM 8401 cells, and measured cell viability and apoptosis by MTT assay and flow cytometry. By western blotting, we evaluated activation of caspase-3, dephosphorylation of IKK, Akt, FOXO1 with time, and change of IKK, Akt, and FOXO1 phosphorylation after transfection of WT IKK and Akt.

Results

Zerumbone (10∽50 μM) induced death of GBM8401 cells in a dose-dependent manner. Flow cytometry studies showed that zerumbone increased the percentage of apoptotic GBM cells. Zerumbone also caused caspase-3 activation and poly (ADP-ribose) polymerase (PARP) production. N-benzyloxycarbonyl -Val-Ala-Asp- fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, hindered zerumbone-induced cell death. Transfection of GBM 8401 cells with WT IKKα inhibited zerumbone-induced apoptosis, and zerumbone significantly decreased IKKα phosphorylation levels in a time-dependent manner. Similarly, transfection of GBM8401 cells with Akt suppressed zerumbone-induced apoptosis, and zerumbone also diminished Akt phosphorylation levels remarkably and time-dependently. Moreover, transfection of GBM8401 cells with WT IKKα reduced the zerumbone-induced decrease in Akt and FOXO1 phosphorylation. However, transfection with WT Akt decreased FOXO1, but not IKKα, phosphorylation.

Conclusion

The results suggest that inactivation of IKKα, followed by Akt and FOXO1 phosphorylation and caspase-3 activation, contributes to zerumbone-induced GBM cell apoptosis.     

Control of the intracellular redox state by glucose participates in the insulin secretion mechanism

Background

Production of reactive oxygen species (ROS) due to chronic exposure to glucose has been associated with impaired beta cell function and diabetes. However, physiologically, beta cells are well equipped to deal with episodic glucose loads, to which they respond with a fine tuned glucose-stimulated insulin secretion (GSIS). In the present study, a systematic investigation in rat pancreatic islets about the changes in the redox environment induced by acute exposure to glucose was carried out.

Methodology/Principal Findings

Short term incubations were performed in isolated rat pancreatic islets. Glucose dose- and time-dependently reduced the intracellular ROS content in pancreatic islets as assayed by fluorescence in a confocal microscope. This decrease was due to activation of pentose-phosphate pathway (PPP). Inhibition of PPP blunted the redox control as well as GSIS in a dose-dependent manner. The addition of low doses of ROS scavengers at high glucose concentration acutely improved beta cell function. The ROS scavenger N-acetyl-L-cysteine increased the intracellular calcium response to glucose that was associated with a small decrease in ROS content. Additionally, the presence of the hydrogen peroxide-specific scavenger catalase, in its membrane-permeable form, nearly doubled glucose metabolism. Interestingly, though an increase in GSIS was also observed, this did not match the effect on glucose metabolism.

Conclusions

The control of ROS content via PPP activation by glucose importantly contributes to the mechanisms that couple the glucose stimulus to insulin secretion. Moreover, we identified intracellular hydrogen peroxide as an inhibitor of glucose metabolism intrinsic to rat pancreatic islets. These findings suggest that the intracellular adjustment of the redox environment by glucose plays an important role in the mechanism of GSIS.     

Mst1-FoxO Signaling Protects Na?ve T Lymphocytes from Cellular Oxidative Stress in Mice

Background

The Ste-20 family kinase Hippo restricts cell proliferation and promotes apoptosis for proper organ development in Drosophila. In C. elegans, Hippo homolog also regulates longevity. The mammalian Ste20-like protein kinase, Mst1, plays a role in apoptosis induced by various types of apoptotic stress. Mst1 also regulates peripheral naïve T cell trafficking and proliferation in mice. However, its functions in mammals are not fully understood.

Methodology/Principal Findings

Here, we report that the Mst1-FoxO signaling pathway plays a crucial role in survival, but not apoptosis, of naïve T cells. In Mst1^−/− mice, peripheral T cells showed impaired FoxO1/3 activation and decreased FoxO protein levels. Consistently, the FoxO targets, Sod2 and catalase, were significantly down-regulated in Mst1^−/− T cells, thereby resulting in elevated levels of intracellular reactive oxygen species (ROS) and induction of apoptosis. Expression of constitutively active FoxO3a restored Mst1^−/− T cell survival. Crossing Mst1 transgenic mice (Mst1 Tg) with Mst1^−/− mice reduced ROS levels and restored normal numbers of peripheral naïve T cells in Mst1 Tg;Mst1^−/− progeny. Interestingly, peripheral T cells from Mst1^−/− mice were hypersensitive to γ-irradiation and paraquat-induced oxidative stresses, whereas those from Mst1 Tg mice were resistant.

Conclusions/Significance

These data support the hypothesis that tolerance to increased levels of intracellular ROS provided by the Mst1-FoxOs signaling pathway is crucial for the maintenance of naïve T cell homeostasis in the periphery.     

GRP78 knockdown enhances apoptosis via the down-regulation of oxidative stress and Akt pathway after epirubicin treatment in colon cancer DLD-1 cells

Introduction

The 78-kDa glucose-regulated protein (GRP78) is induced in the cancer microenvironment and can be considered as a novel predictor of responsiveness to chemotherapy in many cancers. In this study, we found that intracellular reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were higher in GRP78 knockdown DLD-1 colon cancer cells compared with scrambled control cells.

Methodology/Principal Findings

Treatment with epirubicin in GRP78 knockdown DLD-1 cells enhanced apoptosis and was associated with decreased production of intracellular ROS. In addition, apoptosis was increased by the antioxidants propyl gallate (PG) and dithiothreitol (DTT) in epirubicin-treated scrambled control cells. Epirubicin-treated GRP78 knockdown cells resulted in more inactivated Akt pathway members, such as phosphorylated Akt and GSK-3β, as well as downstream targets of β-catenin expression. Knockdown of Nrf2 with small interfering RNA (siRNA) increased apoptosis in epirubicin-treated GRP78 knockdown cells, which suggested that Nrf2 may be a primary defense mechanism in GRP78 knockdown cells. We also demonstrated that epirubicin-treated GRP78 knockdown cells could decrease survival pathway signaling through the redox activation of protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase that negatively regulates the Akt pathway.

Conclusions

Our results indicate that epirubicin decreased the intracellular ROS in GRP78 knockdown cells, which decreased survival signaling through both the Akt pathway and the activation of PP2A. Together, these mechanisms contributed to the enhanced level of epirubicin-induced apoptosis that was observed in the GRP78 knockdown cells.     

Hyperforin inhibits Akt1 kinase activity and promotes caspase-mediated apoptosis involving Bad and Noxa activation in human myeloid tumor cells

Background

The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo.

Methodology and Results

HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser⁴⁷³) and Akt1 substrate Bad (at Ser¹³⁶) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells.

Significance

Our data provide new evidence that HF''s pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment.     

Mechanisms of Siglec-F-Induced Eosinophil Apoptosis: A Role for Caspases but Not for SHP-1, Src Kinases,NADPH Oxidase or Reactive Oxygen

Background

Siglec-F and Siglec-8 are functional paralog proapoptotic cell surface receptors expressed on mouse and human eosinophils, respectively. Whereas Siglec-8 mediated death involves caspases and/or reactive oxygen species (ROS) generation and mitochondrial injury, very little is known about Siglec-F-mediated signaling and apoptosis. Therefore the objective of the current experiments was to better define apoptosis pathways mediated by Siglec-F and Siglec-8. Given that Siglec-F-induced apoptosis is much less robust than Siglec-8-induced apoptosis, we hypothesized that mechanisms involved in cell death via these receptors would differ.

Methods

Consequences of engagement of Siglec-F on mouse eosinophils were studied by measuring ROS production, and by performing apoptosis assays using eosinophils from normal, hypereosinophilic, NADPH oxidase-deficient, src homology domain-containing protein tyrosine phosphatase (SHP)-1-deficient, and Lyn kinase-deficient mice. Inhibitors of caspase and Src family kinase activity were also used.

Results

Engagement of Siglec-F induced mouse eosinophil apoptosis that was modest in magnitude and dependent on caspase activity. There was no detectable ROS generation, or any role for ROS, NADPH oxidase, SHP-1, or Src family kinases in this apoptotic process.

Conclusions

These data suggest that Siglec-F-mediated apoptosis is different in both magnitude and mechanisms when compared to published data on Siglec-8-mediated human eosinophil apoptosis. One likely implication of this work is that models targeting Siglec-F in vivo in mice may not provide identical mechanistic predictions for consequences of Siglec-8 targeting in vivo in humans.     

The Mechanism of Intralipid?-Mediated Cardioprotection Complex IV Inhibition by the Active Metabolite,Palmitoylcarnitine, Generates Reactive Oxygen Species and Activates Reperfusion Injury Salvage Kinases

Background

Intralipid® administration at reperfusion elicits protection against myocardial ischemia-reperfusion injury. However, the underlying mechanisms are not fully understood.

Methods

Sprague-Dawley rat hearts were exposed to 15 min of ischemia and 30 min of reperfusion in the absence or presence of Intralipid® 1% administered at the onset of reperfusion. In separate experiments, the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)-glycine was added either alone or with Intralipid®. Left ventricular work and activation of Akt, STAT3, and ERK1/2 were used to evaluate cardioprotection. ROS production was assessed by measuring the loss of aconitase activity and the release of hydrogen peroxide using Amplex Red. Electron transport chain complex activities and proton leak were measured by high-resolution respirometry in permeabilized cardiac fibers. Titration experiments using the fatty acid intermediates of Intralipid® palmitoyl-, oleoyl- and linoleoylcarnitine served to determine concentration-dependent inhibition of complex IV activity and mitochondrial ROS release.

Results

Intralipid® enhanced postischemic recovery and activated Akt and Erk1/2, effects that were abolished by the ROS scavenger N-(2-mercaptopropionyl)glycine. Palmitoylcarnitine and linoleoylcarnitine, but not oleoylcarnitine concentration-dependently inhibited complex IV. Only palmitoylcarnitine reached high tissue concentrations during early reperfusion and generated significant ROS by complex IV inhibition. Palmitoylcarnitine (1 µM), administered at reperfusion, also fully mimicked Intralipid®-mediated protection in an N-(2-mercaptopropionyl)-glycine -dependent manner.

Conclusions

Our data describe a new mechanism of postconditioning cardioprotection by the clinically available fat emulsion, Intralipid®. Protection is elicited by the fatty acid intermediate palmitoylcarnitine, and involves inhibition of complex IV, an increase in ROS production and activation of the RISK pathway.     

α-Lipoic Acid Reduces Infarct Size and Preserves Cardiac Function in Rat Myocardial Ischemia/Reperfusion Injury through Activation of PI3K/Akt/Nrf2 Pathway

Background

The present study investigates the effects and mechanisms of α-Lipoic acid (LA) on myocardial infarct size, cardiac function and cardiomyocyte apoptosis in rat hearts subjected to in vivo myocardial ischemia/reperfusion (MI/R) injury.

Methodology/Principal Findings

Male adult rats underwent 30 minutes of ischemia followed by 3, 24, or 72 h of reperfusion. Animals were pretreated with LA or vehicle before coronary artery ligation. The level of MI/R- induced LDH and CK release, infarct size, cardiomyocyte apoptosis and cardiac functional impairment were examined and compared. Western blot analysis was performed to elucidate the mechanism of LA pretreatment. The level of inflammatory cytokine TNF-α released to serum and accumulated in injured myocardium as well as neutrophil accumulation in injured myocardium were also examined after MI/R injury. Our results reveal that LA administration significantly reduced LDH and CK release, attenuated myocardial infarct size, decreased cardiomyocytes apoptosis, and partially preserved heart function. Western blot analysis showed that LA pretreatment up-regulated Akt phosphorylation and Nrf2 nuclear translocation while producing no impact on p38MAPK activation or nitric oxide (NO) production. LA pretreatment also increased expression of HO-1, a major target of Nrf2. LA treatment inhibited neutrophil accumulation and release of TNF-α. Moreover, PI3K inhibition abolished the beneficial effects of LA.

Conclusions/Significance

This study indicates that LA attenuates cardiac dysfunction by reducing cardiomyoctyes necrosis, apoptosis and inflammation after MI/R. LA exerts its action by activating the PI3K/Akt pathway as well as subsequent Nrf2 nuclear translocation and induction of cytoprotective genes such as HO-1.