Protective effect of ligustrazine against myocardial ischaemia reperfusion in rats: the role of endothelial nitric oxide synthase

1. The aim of the present study was to determine whether ligustrazine (2,3,5,6-tetramethylpyrazine; TMP) exerts a cardioprotective effect during myocardial ischaemia reperfusion (IR), and to investigate the underlying mechanisms and the role of endothelial nitric oxide synthase (eNOS) in cardioprotection. 2. Sprague-Dawley rats were divided into a sham group and five IR groups: IR control, TMP pretreated, TMP + wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor), N(G) -nitro-L-arginine methyl ester (L-NAME; a NOS inhibitor) and TMP + L-NAME. IR was produced by 35 min of regional ischaemia followed by 120 min of reperfusion. Myocardial infarct size, oxidative stress, myocardial apoptosis, nitric oxide (NO) production, and expression of phosphorylated protein kinase B (Akt) and eNOS were measured. 3. TMP markedly decreased infarct size and attenuated myocardial apoptosis, as evidenced by a decrease in the apoptotic index and reduced caspase-3 activity. TMP treatment caused a marked increase in NO production. Cotreatment with wortmannin or L-NAME completely blocked the TMP-induced NO increase. TMP induced phosphorylation of Akt at Ser 473 (1.61 ± 0.18 vs 0.79 ± 0.10 in the IR control group) and phosphorylation of eNOS at Ser1177 (1.87 ± 0.33 vs 0.94 ± 0.22 in the IR control group). Wortmannin abrogated the phosphorylation of Akt and eNOS induced by TMP. 4. These data suggest that ligustrazine has anti-apoptotic and cardioprotective effects against myocardial IR injury and that it acts through the PI3K/Akt pathway. In addition, the phosphorylation of eNOS with subsequent NO production was found to be an important downstream effector that contributes significantly to the cardioprotective effect of TMP.     

Protective role of PI3-kinase-Akt-eNOS signalling pathway in intestinal injury associated with splanchnic artery occlusion shock

BACKGROUND AND PURPOSE: Endothelial NO synthase (eNOS) is a dynamic enzyme tightly controlled by co- and post-translational lipid modifications, phosphorylation and regulated by protein-protein interactions. Here we have pharmacologically modulated the activation of eNOS, at different post-translational levels, to assess the role of eNOS-derived NO and of these regulatory mechanisms in intestinal injury associated with splanchnic artery occlusion (SAO) shock. EXPERIMENTAL APPROACH: SAO shock was induced by clamping both the superior mesenteric artery and the celiac trunk for 45 min followed by 30 min of reperfusion. During ischemia, 15 min prior to reperfusion, mice were given geldanamycin, an inhibitor of hsp90 recruitment to eNOS, or LY-294002 an inhibitor of phosphatidylinositol 3-kinase (PI3K), an enzyme that initiates Akt-catalysed phosphorylation of eNOS on Ser1179. After 30 min of reperfusion, samples of ileum were taken for histological examination or for biochemical studies. KEY RESULTS: Either LY-294002 or geldanamycin reversed the increased activation of eNOS and Akt observed following SAO shock. These molecular effects were mirrored in vivo by an exacerbation of the intestinal damage. Histological damage also correlated with neutrophil infiltration, assessed as myeloperoxidase activity, and with an increased expression of the adhesion proteins: ICAM-I, VCAM, P-selectin and E-selectin. CONCLUSIONS AND IMPLICATIONS: Overall these results suggest that activation of the Akt pathway in ischemic regions of reperfused ileum is a protective event, triggered in order to protect the intestinal tissue from damage induced by ischaemia/reperfusion through a fine tuning of the endothelial NO pathway.     

二氮嗪后处理对缺血/再灌注心肌的保护作用及其与PI3K/Akt信号通路的关系

目的研究PI3K/Akt信号通路是否参与二氮嗪后处理减轻大鼠心肌缺血/再灌注(I/R)损伤。方法 60只♂SD大鼠随机分为5组(n=12):假手术组(S组)、I/R组、二氮嗪(D组)、wortmannin组(W组)、二氮嗪+wortmannin组(D+W组)。建立大鼠在体心脏I/R模型,除S组外,其余各组均缺血30 min,再灌注120 min。再灌注前5 min,5组分别依次经股静脉输注0.1%DMSO、0.1%DMSO、二氮嗪7mg.kg-1、wortmannin 15μg.kg-1和二氮嗪7 mg.kg-1,其中D+W组于给予二氮嗪前5 min输注wortmannin 15μg.kg-1。记录缺血前、缺血30 min和再灌注120 min时心率(HR)、左室发展压(LVDP)、左室舒张末压(LVEDP);再灌注末,2,3,5-氯化三苯基四氮唑(TTC)染色法检测心肌梗死面积、TUNEL染色法检测心肌细胞凋亡率、Western blot分析p-Akt表达水平。结果各组缺血前心功能指标HR、LVDP、LVEDP无差异(P>0.05)。再灌注120 min后,与I/R组相比,D组和D+W组LVDP明显升高、LVEDP明显降低(P<0.01或<0.05),梗死面积与凋亡率降低(P<0.01或0.05),D组p-Akt表达水平升高(P<0.01);与D组比,D+W组LVDP降低(P<0.05),梗死面积与凋亡率增高(P<0.05),p-Akt表达水平降低(P<0.01)。结论二氮嗪后处理部分通过激活PI3K/Akt信号通路减轻大鼠在体心肌I/R损伤。     

Acute reduction of myocardial infarct size by a hydroxymethyl glutaryl coenzyme A reductase inhibitor is mediated by endothelial nitric oxide synthase

In addition to their lipid-lowering properties, statins improve endothelial function by increasing the activity of endothelial nitric oxide synthase (eNOS). It was hypothesized that, by this mechanism, statins protect the myocardium from ischemia/reperfusion injury in normocholesterolemic animals. Rats were pretreated for 1 week with either cerivastatin (0.3 mg/kg/d) or placebo. Anesthetized animals underwent 30 minutes of coronary artery occlusion (CAO) followed by 180 minutes of reperfusion. In a separate set of experiments, the NOS inhibitor l-NAME (15 mg/kg; N -nitro-l-arginine methyl ester) was administered 15 minutes before CAO. Cerivastatin decreased infarct size by 49% (P < 0.05) without reducing plasma cholesterol levels. Cerivastatin increased myocardial eNOS mRNA and NOS activity and by 52% and 58% (P < 0.05), respectively. Cardioprotection and upregulation of eNOS activity evoked by cerivastatin were not observed in rats cotreated with l-NAME. These results show that statins reduce the extent of myocardial necrosis in normocholesterolemic rats after acute ischemia/reperfusion injury by increasing myocardial eNOS activity. Therefore, statins may protect the heart not only by reducing the incidence of ischemic events, but also by limiting cell damage during acute myocardial infarction.     

Glimepiride Treatment Upon Reperfusion Limits Infarct Size via the phosphatidylinositol 3-Kinase/Akt Pathway in Rabbit Hearts

The phenomenon termed postconditioning, that is, brief episodes of ischemia/reperfusion at the onset of reperfusion reduce infarct size, is thought to involve the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Treatment with a drug activating PI3K at the onset of reperfusion may confer a similar cardioprotection. The sulfonylurea glimepiride has been shown to activate PI3K in human endothelial cells. We therefore tested in rabbit hearts whether glimepiride can produce postconditioning-mimetic actions. Langendorff-perfused rabbit hearts were subjected to 30 min of global ischemia and 120 min of reperfusion, and infarct size was determined by triphenyltetrazolium staining. Phosphorylation of Akt was analyzed by Western blotting. Glimepiride (10 μM) treatment for the first 10 min of reperfusion significantly reduced infarct size from 67.2 ± 1.3% in controls to 35.8 ± 4.5% (P<0.01). This infarct size–limiting effect of glimepiride was abolished by a selective inhibitor of PI3K (5 μM LY294002, 65.4 ± 3.4%). Phosphorylation of the PI3K substrate Akt was significantly increased in glimepiride-treated hearts when compared to controls (P<0.05). Glimepiride-induced Akt phosphorylation was inhibited by LY294002. In conclusion, our study demonstrates that glimepiride treatment upon reperfusion reduces infarct size in rabbit hearts via a PI3K/Akt-mediated pathway. The postconditioning-mimetic action of glimepiride may be beneficial for the treatment of diabetic patients with ischemic heart disease.     

17-methoxyl-7-hydroxyl-benzofuran chalcone alleviates myocardial ischemia/reperfusion injury via PI3K/Akt signal pathway北大核心CSCD

Aim To investigate the effects of 17-me-thoxyl-7-hydroxyl-benzofuran chalcone ( YLSC ) on myocardial ischemia/reperfusion injury ( MI/RI ) by modulating PI3K/Akt signaling pathway and the possible mechanisms. Methods Male SD rats were randomly divided into sham group, model group, YLSC group, wortmannin(WM) group and YLSC + WM group (n = 8 ). The rat model of MI/RI was established by ligation of the left anterior descending artery for 30 min followed by loosening the ligature for 2 h. After reperfusion, blood samples were obtained to determine serum contents of CK-MB, LDH, NO and TNF-ot. The protein levels of total (t) -Akt, phosphorylated (p) -Akt and LC3-U. were detected by Western blot. Caspase-3, Beclinl and eNOS niRNA expression was evaluated by FQ-PCR. Results YLSC pretreatment greatly reduced serum levels of CK-MB, LDH and TNF-α, and elevated NO content. It also inhibited the expression of caspase-3, Beclinl and LC3-II, while enhanced p-Akt and eNOS expression. Conclusion YLSC protects the heart against MI/RI via inhibition of apoptosis and excessive autophagy, in which protective effect is regulated by activation of the PI3K/Akt pathway.     

Luteolin inhibits apoptosis and improves cardiomyocyte contractile function through the PI3K/Akt pathway in simulated ischemia/reperfusion

Luteolin, a naturally occurring polyphenol flavonoid, has demonstrated to exert myocardial protection effects. However, the mechanisms have not been fully elucidated. In the present study, we investigated whether luteolin pretreatment was associated with cardioprotection in a rat ischemia/reperfusion (I/R) model. Luteolin significantly not only restored contractility of the left ventricle, but also reduced the infarct size and lactate dehydrogenase leakage during I/R. In addition, luteolin pretreatment significantly improved cardiomyocyte shortening amplitude, decreased the apoptotic rate, upregulated Bcl-2 expression, downregulated Bax expression and raised the Bcl-2/Bax ratio under a simulated ischemia/reperfusion (SI/R) condition. Moreover, luteolin pretreatment increased protein kinase B (Akt) phosphorylation, phospholamban phosphorylation and the expression of sarcoplasmic reticulum calcium ATPase following SI/R. The phosphoinositide 3-kinase (PI3K)/Akt pathway is one of the most important intracellular survival signal pathways. To determine whether luteolin-induced cardioprotection was mediated by the PI3K/Akt pathway, we utilized the PI3K inhibitor LY294002. Inhibition of Akt activity markedly abolished luteolin-induced positive contraction and inhibition of apoptosis in SI/R cardiomyocytes. These results showed that luteolin inhibits apoptosis and improves cardiomyocyte contractile function at least partly through the PI3K/Akt pathway in SI/R.     

PI3K/Akt调节线粒体Cx43蛋白表达在H_2S后处理离体大鼠心肌中的保护作用

目的探讨PI3K/Akt信号通路是否通过调节线粒体缝隙连接蛋白Cx43而在硫化氢(H2S)后处理中减轻离体大鼠心脏缺血/再灌注(I/R)损伤。方法 56只♂SpragueDawley(SD)大鼠随机分为4组(n=14):缺血/再灌注组(I/R组),PI3K/Akt信号通路抑制剂LY294002组(LY组),硫化氢后处理组(NP组),硫化氢后处理+PI3K/Akt信号通路抑制剂LY294002组(N+L组)。采用离体心脏Langen-dorff灌注模型,平衡灌注20 min后停灌30 min复灌60 min。记录平衡末及灌注结束时的心率(HR)、左室舒张末期压(LVEDP)、左室发展压(LVDP)、左室内压上升最大速率(+dp/dtmax)、左室内压下降最大速率(-dp/dtmax);灌注结束时,TTC法染色心肌切片并计算心肌梗死面积百分比;TUNEL法检测心肌细胞凋亡,计算凋亡指数(AI);Westernblot半定量线粒体总的Cx43(total connexin 43,tCx43)和磷酸化Cx43(phosphorylated connexin 43,pCx43)表达水平。结果平衡灌注末各组间心功能指标差异无统计学意义。再灌注后,与I/R组比较,NP组心功能的各项指标明显改善(P<0.05);心肌梗死面积减少(26.5±4.2)%vs(44.5±5.3)%(P<0.05);凋亡指数降低(25.9±3.0)%vs(43.1±1.9)%(P<0.05);线粒体tCx43和pCx43蛋白表达水平明显升高。LY294002逆转了H2S后处理产生的心肌保护效应,使N+L组心功能指标及线粒体中tCx43和pCx43的表达水平降低(P<0.05),心肌梗死面积及凋亡指数均增加(P<0.05)。结论 PI3K/Akt信号通路通过上调线粒体缝隙连接蛋白Cx43蛋白的表达而在硫化氢(H2S)后处理中减轻离体大鼠I/R损伤。     

Water extract of Korean red ginseng stimulates angiogenesis by activating the PI3K/Akt-dependent ERK1/2 and eNOS pathways in human umbilical vein endothelial cells

Angiogenesis is important for promoting cardiovascular disease, wound healing, and tissue regeneration. We investigated the effects of Korean red ginseng water extract (KRGE) on angiogenesis and its underlying signal mechanism. KRGE increased in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells, as well as stimulated in vivo angiogenesis without increasing VEGF expression. KRGE-induced angiogenesis was accompanied by phosphorylation of ERK1/2, phosphatidylinositol 3-kinase (Akt), and endothelial nitric oxide synthase (eNOS) as well as an increase in NO production. Inhibition of PI3K activity by wortmannin completely inhibited KRGE-induced angiogenesis and phosphorylation of Akt, ERK1/2, and eNOS, indicating that PI3K/Akt activation is an upstream event of the KRGE-mediated angiogenic pathway. The MEK inhibitor PD98059 blocked KRGE-induced ERK1/2 phosphorylation without affecting Akt and eNOS activation. However, the eNOS inhibitor N(G)-monomethyl-L-arginine effectively inhibited tube formation, but partially blocked proliferation and migration as well as ERK phosphorylation, without altering Akt and eNOS activation, revealing that the eNOS/NO pathway is partially involved in ERK1/2 activation. This study demonstrated that KRGE stimulates in vitro and in vivo angiogenesis through the activation of the PI3K/Akt-dependent ERK1/2 and eNOS signal pathways and their cross talk.     

Epigallocatechin-3-gallate,a green tea catechin,protects the heart against regional ischemia–reperfusion injuries through activation of RISK survival pathways in rats

Epigallocatechin-3-gallate (EGCG), the major catechin derived from green tea, has been shown to modulate numerous molecular targets in the setting of inflammation. This study aimed to determine whether EGCG protects against regional myocardial ischemia/reperfusion (I/R) injuries and its underlying mechanisms involving the role of reperfusion injury salvage kinase (RISK) pathways (PI3K-Akt and ERK 1/2) and GSK-3β or apoptotic kinases (p38 and JNK). The rats were subjected to I/R injuries consisting of 30 min ischemia followed by 2 h reperfusion. EGCG (10 mg/kg, intravenously) was administered alone or along with wortmannin (PI3K inhibitor, 0.6 mg/kg, intravenously) 5 min before the onset of reperfusion. Wortmannin was administered 10 min before the reperfusion. Infarct size was measured at the end of the reperfusion. The phosphorylation of Akt, GSK-3β, and MAPK kinases (ERK1/2, P38 and JNK) was determined by Western blotting after 10 min of reperfusion. EGCG reduced the infarct size compared with the control (25.4 ± 9.2 versus 43.2 ± 8.2 %, p < 0.05). Wortmannin alone did not affect the infarct size, but abolished the EGCG-induced infarct size limiting effect, indicating that EGCG may protect the heart by modulating the PI3K-Akt. EGCG significantly enhanced the phosphorylation of Akt and GSK-3β but not ERK1/2, while it reduced that of p38 and JNK. These results suggest that EGCG has a protective effect against regional myocardial I/R injuries through activation of the RISK pathway and attenuation of p38 and JNK. EGCG may have cardioprotective effects in patients undergoing surgeries prone to myocardial I/R injuries.     

Neuroprotection of selenite against ischemic brain injury through negatively regulating early activation of ASK1/JNK cascade via activation of PI3K/AKT pathway

AIM: To investigate whether selenite, a known antioxidant, could decrease the activation of apoptosis signal regulating kinase 1/c-jun N-terminal kinase (ASK1/ JNK) signaling cascade in cerebral ischemia/reperfusion (I/R) by activating the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in rat hippocampi, and the neuroprotective effect of selenite against ischemic injury after 15 min of transient brain ischemia. METHODS: Transient global brain ischemia was induced by 4-vessel occlusion into adult male Sprague-Dawley rats weighing 250-300 g. The rats were pretreated only with selenite (0.3 mg/kg dissolved in 0.9% saline) every 24 h for 7 d by means of intravenous injection of the tail or combined with LY294002 from d 5 by left cerebral ventricle injection before surgery. RESULTS: Selenite significantly increased AKT1 activation and decreased the activation of ASK1/ JNK cascade via phosphorylating ASK1 at Ser-83 residue by AKT1 during early reperfusion after 15 min transient global brain ischemia. On the contrary, combined pretreatment of the rats with LY294002 (a specific PI3K inhibitor) and selenite significantly inhibited the effects solely with selenite. CONCLUSION: The activation of the pro-apoptotic ASK1/JNK cascade, which is closely associated with oxidative stress, could be suppressed by selenite through activating the antiapoptotic PI3K/AKT pathway during early reperfusion after cerebral ischemia in rat hippocampi.     

苦参碱的心脏保护作用及其机制的研究进展

苦参碱对心脏具有正性肌力、负性频率和负性自律性的作用,因此具有广谱的抗心律失常作用。苦参碱对冠脉结扎缺血、缺血再灌注、异丙肾上腺素、高血脂、糖尿病和阿霉素引起的心肌损伤都有保护作用。苦参碱还能阻滞心肌肥大和纤维化的发生、发展。苦参碱保护心脏作用的基本机制是抗氧化、抗炎、降血糖、调血脂作用,由此抑制ROS/TLR4信号通路、PI3K/Akt信号通路和TGF-β诱导的PERK信号通路,激活Akt和STAT3信号通路和AMPKα/UCP2信号通路,上调JAK2/STAT3通路上的HSP70以及JNK、ASK1、e NOS和一氧化氮的表达,提高线粒体ATP酶活性,保护线粒体而减少心肌细胞凋亡、减轻心肌损伤和抑制胶原合成,从而抑制心肌肥大和纤维化。从抗各种心肌缺血性损伤、抗阿霉素诱导的心肌损伤、抗高糖性心肌损伤、抗心肌肥大和纤维化4个方面综述苦参碱的心脏保护作用及其机制。     

Rosiglitazone‐induced myocardial protection against ischaemia–reperfusion injury is mediated via a phosphatidylinositol 3‐kinase/Akt‐dependent pathway

1. Rosiglitazone is widely used in the treatment of Type 2 diabetes. However, in recent years it has become evident that the therapeutic effects of peroxisome proliferator‐activated receptor γ ligands reach far beyond their use as insulin sensitizers. Recently, the ability of rosiglitazone pretreatment to induce cardioprotection following ischaemia–reperfusion (I/R) has been well documented; however, the protective mechanisms have not been elucidated. In the present study, examined the role of the phosphatidylinositol 3‐kinase (PI3‐K)/Akt signalling pathway in rosiglitazone cardioprotection following I/R injury. 2. Mice were pretreated with 3 mg/kg per day rosiglitazone for 14 days before hearts were subjected to ischaemia (30 min) and reperfusion (2 h). Wortmannin (1.4 mg/kg, i.p.), an inhibitor of PI3‐K, was administered 10 min prior to myocardial I/R. Then, activation of the PI3‐K/Akt/glycogen synthase kinase (GSK)‐3α signalling pathway was examined. The effects of PI3‐K inhibition on rosiglitazone‐induced cardioprotection were also evaluated. 3. Compared with control rats, the ratio of infarct size to ischaemic area (area at risk) and the occurrence of sustained ventricular fibrillation in rosiglitazone‐pretreated rats was significantly reduced (P < 0.05). Rosiglitazone pretreatment attenuated cardiac apoptosis, as assessed by ELISA to determine cardiomyocyte DNA fragmentation. Rosiglitazone pretreatment significantly increased levels of phosphorylated (p‐) Akt and p‐GSK‐3α in the rat myocardium. Pharmacological inhibition of PI3‐K by wortmannin markedly abolished the cardioprotection induced by rosiglitazone. 4. These results indicate that rosiglitazone‐induced cardioprotection in I/R injury is mediated via a PI3‐K/Akt/GSK‐3α‐dependent pathway. The data also suggest that modulation of PI3‐K/Akt/GSK‐3α‐dependent signalling pathways may be a viable strategy to reduce myocardial I/R injury.     

Urantide对大鼠心肌缺血/再灌注后心肌细胞凋亡的作用及机制研究

目的观察urantide对大鼠缺血/再灌注心肌组织氧化应激损伤的作用和心肌细胞凋亡的影响及其与PI3K/Akt及PKC信号通路的关系。方法可逆性冠脉左前降支结扎造成心肌缺血/再灌注模型,给予大鼠心脏缺血30 min再灌注90 min。80只大鼠随机分为假手术组、缺血/再灌注(I/R)组、urantide低、中、高剂量组(3,10,30μg.kg-1)、维拉帕米对照组(1.6 mg.kg-1)、urantide+CHE组(30μg.kg-1+1 mg.kg-1)、urantide+LY294002组(30μg.kg-1+0.3 mg.kg-1)。Urantide低、中、高剂量组中urantide于缺血前5 min舌下静脉1 min内一次性推注,urantide+CHE组与urantide+LY294002组中,在穿线稳定后舌下静脉分别快速推注CHE与LY294002,5 min后舌下静脉快速推注uran-tide 30μg.kg-1,稳定10 min后再行缺血/再灌注操作。实验结束后测定大鼠血清中超氧化物歧化酶(SOD)、一氧化氮合酶(NOS)活力和丙二醛(MDA)含量以及一氧化氮(NO)含量。TUNEL法检测凋亡细胞指数(AI),免疫组化法检测心肌组织Bcl-2、Bax的蛋白表达。结果与I/R组相比,urantide 30μg.kg-1能明显升高SOD活性(P<0.01),明显减少血清中MDA的含量(P<0.05),明显提高NOS活力(P<0.01),使NO含量增加(P<0.01);Bax蛋白表达和心肌细胞凋亡指数降低(P<0.01),Bcl-2蛋白表达增加(P<0.05);urantide+CHE组与urantide+LY294002组与urantide30μg.kg-1组相比,SOD活力和NO含量下降,MDA含量上升,心肌组织中Bax蛋白表达和心肌细胞凋亡指数上升,而Bcl-2蛋白表达下降,与I/R组比较差异无统计学意义(P>0.01)。结论 Urantide能够通过激活PKC和PI3K/Akt信号转导通路,减少心肌组织氧自由基的含量,进一步调控Bcl-2和Bax蛋白的表达,抑制心肌缺血/再灌注大鼠心肌细胞的凋亡,从而对大鼠心肌缺血/再灌注具有一定保护作用。     

PTEN, the Achilles' heel of myocardial ischaemia/reperfusion injury?

Myocardial ischaemia/reperfusion injury leading to myocardial infarction is one of the most frequent causes of debilitation and death in man. Considerable research has been undertaken to investigate the possibility of reducing myocardial infarction and increasing cell survival by activating certain endogenous prosurvival signaling pathways. Thus, it has been established that the activation of the PI3K (Phosphoinositide-3 kinase)/Akt (Protein kinase B, PKB) signaling pathway is essential for protection against ischaemia/reperfusion injury. This pathway has been shown to be activated by mechanical procedures (e.g. pre and post conditioning) as well as by a number of pharmacological agents. Although the activation of this prosurvival signaling pathway induces the phosphorylation of a large number of substrates implicated in increased cell survival, when activated over a prolonged period this pathway can have detrimental consequences by facilitating unwanted growth and malignancies. Importantly PTEN (phosphatase and tensin homolog deleted on chromosome ten), is the main phosphatase which negatively regulates the PI3K/Akt pathway. In this review we discuss: a) the significance and the limitations of inhibiting PTEN in myocardial ischaemia/reperfusion injury; b) PTEN and its relationship to ischaemic preconditioning, c) the role of PTEN in the development of tolerance to chronic administration of drugs known to limit infarction by activating PI3K/Akt pathway when given acutely, and d) the possible role of PTEN in the ischaemic/reperfused diabetic heart. The experimental evidence discussed in this review illustrates the importance of PTEN inhibition in the protection of the heart against ischaemia/reperfusion injury.     

Cardioprotection with adenosine A2 receptor activation at reperfusion

Pre-ischemic treatment is seldom possible in the clinical setting of acute myocardial infarction. Thus, to successfully save myocardium from infarction, it is required that protective interventions must be effective when applied after ischemia has begun or at the onset of reperfusion. Unfortunately, in spite of a large body of experimental data showing that various interventions are cardioprotective at reperfusion, no specific therapy has yet been established to be clinically applicable. However, recent data from several laboratories have shown that adenosine and its analogues given at reperfusion can markedly protect the heart from ischemia/reperfusion injury. While the experimental data suggest that factors such as adenosine A2 receptor activation, anti-neutrophil effect, attenuation of free radical generation, increased nitric oxide (NO) availability, activation of the PI3-kinase/Akt pathway and ERK, prevention of mitochondrial damage, and anti-apoptotic effects may be involved in the protective effect of adenosine or its analogues, the exact receptor subtype(s), the detailed signaling mechanisms, and interaction between those individual factors are still unknown. A definite answer to these unsolved problems will offer insights into the mechanisms of cardioprotection at reperfusion, and will be critical for developing a successful therapeutic strategy to salvage ischemic myocardium in patients with acute myocardial infarction.     

Mechanisms mediating the cardioprotective effects of rapamycin in ischaemia-reperfusion injury

1. In the present study, the temporal and concentration-dependent cardioprotective effects of rapamycin against ischaemia-reperfusion (I/R) injury, as well as the underlying mechanisms, were investigated. 2. Rat Langendorff-perfused isolated hearts were exposed to 40 min global ischaemia followed by 120 min reperfusion. Hearts were perfused with different concentrations of rapamycin before and after ischaemia. Myocardial injury was assessed in terms of infarct size and the release of lactate dehydrogenase (LDH) and creatine kinase (CK). The phosphorylation of Akt, extracellular signal-regulated kinase (ERK) 1/2 and endothelial nitric oxide synthase (eNOS) was determined at the end of reperfusion. 3. When administered prior to ischaemia, 25, 50 and 100 nmol/L rapamycin significantly reduced infarct size compared with control (40.1 ± 1.5, 26.3 ± 4.1 and 21.2 ± 3.4 vs 52.5 ± 4.5%, respectively) without affecting the recovery of ventricular function. No reduction in infarct size was observed when 50 nmol/L rapamycin was administered 10 or 120 min into the reperfusion period. 4. Rapamycin (50 nmol/L) enhanced the phosphorylation of Akt kinase but did not affect the phosphorylation of ERK1/2 or eNOS at the end of reperfusion. The cardioprotective effect of rapamycin was blocked by the phosphatidylinositol 3-kinase (Akt) inhibitor LY294002 (15 nmol/L). 5. In conclusion, rapamycin mediates cardioprotection prior to ischaemia and after reperfusion. This protection may involve activation of the phosphatidylinositol 3-kinase pathway.     

Simvastatin induces a central hypotensive effect via Ras-mediated signalling to cause eNOS up-regulation

BACKGROUND AND PURPOSE

Clinical studies indicate that statins have a BP-lowering effect in hypercholesterolemic individuals with hypertension. Specifically, statins modulate BP through the up-regulation of endothelial NOS (eNOS) activation in the brain. However, the signalling mechanisms through which statins enhance eNOS activation remain unclear. Therefore, we examined the possible signalling pathways involved in statin-mediated BP regulation in the nucleus tractus solitarii (NTS).

EXPERIMENTAL APPROACH

To investigate the involvement of Ras and other signalling pathways in simvastatin-induced effects on BP, BP and renal sympathetic nerve activity (RSNA) were determined in spontaneously hypertensive rats (SHRs) before and after i.c.v. administration of simvastatin in the absence and presence of a Ras-specific inhibitor (farnesyl thiosalicylic acid, FTS), a geranylgeranyltransferase inhibitor (GGTI-2133), a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or a MAPK-ERK kinase (MEK) inhibitor (PD98059).

KEY RESULTS

FTS significantly attenuated the decrease in BP and increased NO evoked by simvastatin and reversed the decrease in basal RSNA induced by simvastatin. Immunoblotting and pharmacological studies showed that inhibition of Ras activity by FTS significantly abolished simvastatin-induced phosphorylation of ERK1/2, ribosomal protein S6 kinase (RSK), Akt and decreased eNOS phosphorylation. Likewise, administration of Akt and ERK1/2 signalling inhibitors, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98059, attenuated the reduction in BP evoked by simvastatin. Furthermore, i.c.v. simvastatin decreased Rac1 activation and the number of ROS-positive cells in the NTS.

CONCLUSIONS AND IMPLICATIONS

Simvastatin modulates central BP control in the NTS of SHRs by increasing Ras-mediated activation of the PI3K-Akt and ERK1/2-RSK signalling pathways, which then up-regulates eNOS activation.     

Alpha-lipoic acid activates eNOS through activation of PI3-kinase/Akt signaling pathway

BackgroundLipoic acid (LA) exerts therapeutic effects on cardiovascular diseases. However, the mechanisms underlying these therapeutic effects remain elusive. Endothelial nitric oxide synthase (eNOS) plays a critical role in cardiovascular homeostasis. LA was shown to potently activate PI3-kinase/Akt pathway, and the latter is critical in the regulation of eNOS activity. In the present study, we test the hypothesis that LA improves endothelial function through PI₃-kinase/Akt-mediated eNOS activation.Methods and ResultsWestern blot analysis showed that LA time- and dose-dependently induced phosphorylation of Akt and eNOS in human umbilical vein endothelial cells (HUVECs). Both PI₃-kinase and Akt inhibitors abolished LA-induced eNOS phosphorylation, indicating that LA induces eNOS phosphorylation through the PI₃-kinase/Akt pathway. This increase in eNOS phosphorylation was paralleled by an increase in NO release by HUVECs, supporting its relevance in eNOS activity regulation. Myograph analysis revealed that LA relaxed phenylephrine-induced contraction. Endothelium removal and NOS inhibition by L-NAME abolished this vasodilator action of LA, and Akt but not AMPK inhibition significantly reduced the vasodilator action of LA, indicating that it is mediated by PI₃-kinase/Akt pathway-dependent activation of eNOS. Consistent with in vitro results, intraperitoneal injection with LA significantly increased plasma nitrite and nitrate levels in C57Bl/6j mice.ConclusionsLA activates eNOS through a PI3-kinase/Akt signaling pathway-dependent mechanism, offering a potential molecular basis for the therapeutic effects of LA on cardiovascular diseases.     

ISCHAEMIC POST-CONDITIONING PROTECTS BOTH ADULT AND AGED SPRAGUE-DAWLEY RAT HEART FROM ISCHAEMIA–REPERFUSION INJURY THROUGH THE PHOSPHATIDYLINOSITOL 3-KINASE–AKT AND GLYCOGEN SYNTHASE KINASE-3β PATHWAYS

• 1 Numerous studies have demonstrated that ischaemic post‐conditioning (IPoC) protects adult rats from myocardial ischaemia‐reperfusion (I/R) injury. Recent evidence suggests compromised cardioprotection by IPoC in aged mice. The present study was designed to test the hypothesis that IPoC protects against I/R injury in aged hearts, potentially through a phosphatidylinositol 3‐kinase (PI3‐K)–Akt‐ and glycogen synthase kinase (GSK)‐3β‐dependent mechanism.
• 2 Hearts from adult (3–4 months) or aged (16–18 months) Sprague‐Dawley rats were subjected in vivo to 30 min ischaemia followed by 3 h reperfusion. Ischaemic post‐conditioning (four cycles of 10 s reperfusion–10 s ischaemia) was applied at the beginning of reperfusion, either alone or in combination with the PI3‐K inhibitor LY294002 (0.3 mg/kg). Infarct size and the phosphorylation of Akt and GSK‐3β were determined.
• 3 Ischaemic post‐conditioning reduced infarct size in both adult and aged rat hearts. This protection was accompanied by a significant increase in phosphorylation of Akt and GSK‐3β. LY294002 abolished the IPoC‐induced phosphorylation of Akt and GSK‐3β, as well as the infarct‐limiting effect of IPoC in adult and aged rats. In addition, IPoC significantly attenuated plasma concentrations of creatine kinase and lactate dehydrogenase after reperfusion in both adult and aged rats.
• 4 In conclusion, IPoC, at the onset of reperfusion, reduces myocardial infarct size in both adult and aged rat hearts, potentially through a PI3‐K‐, Akt‐ and GSK‐3β‐dependent mechanism.