Lovastatin protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis by activation of PI3K/Akt and ERK1/2

Cell therapy with bone marrow-derived mesenchymal stem cells (MSCs) has been shown to have great promises in cardiac repair after myocardial infarction. However, poor viability of transplanted MSCs in the infracted heart has limited the therapeutic efficacy. Our previous studies have shown in vitro that rat MSCs undergo caspase-dependent apoptosis in response to hypoxia and serum deprivation (Hypoxia/SD). Recent findings have implicated statins, an established class of cholesterol-lowering drugs, enhance the survival of cells under various conditions. In this study, we investigated the effect of lovastatin on rat MSCs apoptosis induced by Hypoxia/SD, focusing in particular on regulation of mitochondrial apoptotic pathway and the survival signaling pathways. We demonstrated that lovastatin (0.01-1 microM) remarkably prevented MSCs from Hypoxia/SD-induced apoptosis through inhibition of the mitochondrial apoptotic pathway, leading to attenuation of caspase-3 activation. The loss of mitochondrial membrane potential and cytochrome-c release from mitochondria to cytosol were significantly inhibited by lovastatin. Furthermore, the antiapoptotic effect of lovastatin on mitochondrial apoptotic pathway was effectively abrogated by both PI3K inhibitor, LY294002 and ERK1/2 inhibitor, U0126. The phosphorylations of Akt/GSK3 beta and ERK1/2 stimulated by lovastatin were detected. The activation of ERK1/2 was inhibited by a PI3K inhibitor, LY294002, but U0126, a ERK1/2 inhibitor did not inhibit phosphorylation of Akt and GSK3 beta. These data demonstrate that lovastatin protects MSCs from Hypoxia/SD-induced apoptosis via PI3K/Akt and MEK/ERK1/2 pathways, suggesting that it may prove a useful therapeutic adjunct for transplanting MSCs into damaged heart after myocardial infarction.     

Angiopoietin-Like 4 Confers Resistance to Hypoxia/Serum Deprivation-Induced Apoptosis through PI3K/Akt and ERK1/2 Signaling Pathways in Mesenchymal Stem Cells

Angiopoietin-like 4 (ANGPTL4) is a potential anti-apoptotic agent for various cells. We examined the protective effect of ANGPTL4 on hypoxia/serum deprivation (SD)-induced apoptosis of MSCs, as well as the possible mechanisms. MSCs were obtained from rat bone marrow and cultured in vitro. Apoptosis was induced by hypoxia/SD for up to 24 hr, and assessed by flow cytometry and TUNEL assay. Expression levels of Akt, ERK1/2, focal adhesion kinase (FAK), Src, Bcl-2, Bax, cytochrome C and cleaved caspase-3 were detected by Western blotting. Integrin β1 mRNA was detected by qRT-PCR. Mitochondrial membrane potential was assayed using a membrane-permeable dye. Hypoxia/SD-induced apoptosis was significantly attenuated by recombinant rat ANGPTL4 in a concentration dependent manner. Moreover, ANGPTL4 decreased the hypoxia/SD-induced caspase-3 cleavage and the cytochrome C release, but increased the Bcl-2/Bax ratio and the mitochondrial membrane potential. Decreased expression of integrin β1, the ANGPTL4 receptor was observed during hypoxia/SD conditions, however, such decrease was reversed by ANGPTL4. In addition, ANGPTL4 induced integrin β1-associated FAK and Src phosphorylation, which was blocked by anti-integrin β1 antibody. ANGPTL4 also reversed the hypoxia/SD-induced decrease of Akt and ERK 1/2 phosphorylation, and the effect of ANGPTL4 was abolished by inhibitors of either integrins, ERK1/2, or phosphatidylinositol 3-kinase (PI3K). Blocking integrinβ1, Akt or ERK largely attenuated anti-apoptotic effect of ANGPTL4. ANGPTL4 protects MSCs from hypoxia/SD-induced apoptosis by interacting with integrins to stimulate FAK complex, leading to downstream ERK1/2 and PI3K/Akt signaling pathways and mimicking the pathway in which MSCs contact with the extracellular matrix.     

LPA rescues ER stress‐associated apoptosis in hypoxia and serum deprivation‐stimulated mesenchymal stem cells

Poor viability of transplanted mesenchymal stem cells (MSCs) in the infracted heart has limited their therapeutic efficacy in cardiac repair after myocardial infarction. We previously demonstrated that hypoxia and serum deprivation (hypoxia/SD) induced mitochondria‐dependent apoptosis in MSCs, while lysophosphatidic acid (LPA) could almost completely block this apoptotic process. However, the role of endoplasmic reticulum (ER) stress and its upstream signaling events in hypoxia/SD‐induced MSC apoptosis remain largely unknown. Here we found that hypoxia/SD‐induced MSC apoptosis was associated with ER stress, as shown by the induction of CHOP expression and procaspase‐12 cleavage, while the effects were abrogated by LPA treatment, suggesting ER stress is also a target of LPA. Furthermore, hypoxia/SD induced p38 activation, inhibition of which resulted in decreases of apoptotic cells, procaspase‐12 cleavage and mitochondrial cytochrome c release that function in parallel in MSC apoptosis. Unexpectedly, p38 inhibition enhanced hypoxia/SD‐induced CHOP expression. Interestingly, p38 activation, a common process mediating various biological effects of LPA, was inhibited by LPA in this study, and the regulation of p38 pathway by LPA was dependent on LPA_1/3/Gi/ERK1/2 pathway‐mediated MKP‐1 induction but independent of PI3K/Akt pathway. Collectively, our findings indicate that ER stress is a target of LPA to antagonize hypoxia/SD‐induced MSC apoptosis, and the modulation of mitochondrial and ER stress‐associated apoptotic pathways by LPA is at least partly dependent on LPA_1/3/Gi/ERK/MKP‐1 pathway‐mediated p38 inhibition. This study may provide new anti‐apoptotic targets for elevating the viability of MSCs for therapeutic potential of cardiac repair. J. Cell. Biochem. 111: 811–820, 2010. © 2010 Wiley‐Liss, Inc.     

Berberine reduces ischemia/reperfusion-induced myocardial apoptosis via activating AMPK and PI3K–Akt signaling in diabetic rats

Diabetes increases the risk of cardiovascular diseases. Berberine (BBR), an isoquinoline alkaloid used in Chinese medicine, exerts anti-diabetic effect by lowering blood glucose and regulating lipid metabolism. It has been reported that BBR decreases mortality in patients with chronic congestive heart failure. However, the molecular mechanisms of these beneficial effects are incompletely understood. In the present study, we sought to determine whether BBR exerts cardioprotective effect against ischemia/reperfusion (I/R) injury in diabetic rats and the underlying mechanisms. Male Sprague-Dawley rats were injected with low dose streptozotocin and fed with a high-fat diet for 12 weeks to induce diabetes. The diabetic rats were intragastrically administered with saline or BBR (100, 200 and 400 mg/kg/d) starting from week 9 to 12. At the end of week 12, all rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion. BBR significantly improved the recovery of cardiac systolic/diastolic function and reduced myocardial apoptosis in diabetic rats subjected to myocardial I/R. Furthermore, in cultured neonatal rat cardiomyocytes, BBR (50 μmol/L) reduced hypoxia/reoxygenation-induced myocardial apoptosis, increased Bcl-2/Bax ratio and decreased caspase-3 expression, together with enhanced activation of PI3K–Akt and increased adenosine monophosphate-activated protein kinase (AMPK) and eNOS phosphorylation. Pretreatment with either PI3K/Akt inhibitor wortmannin or AMPK inhibitor Compound C blunted the anti-apoptotic effect of BBR. Our findings demonstrate that BBR exerts anti-apoptotic effect and improves cardiac functional recovery following myocardial I/R via activating AMPK and PI3K–Akt–eNOS signaling in diabetic rats.     

恒磁场抑制缺血缺氧条件下大鼠骨髓间充质干细胞凋亡

目的:研究恒磁场对体外缺血缺氧培养条件下大鼠骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs) 凋亡的影响并探讨其作用机制.方法:采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出骨髓间充质干细胞(MSCs).经形态学和流式细胞仪检测MSCs表面标志物鉴定后,将骨髓间充质干细胞(BMSCs)在缺血缺氧条件下培养,通过TUNEL检测比较不同组别细胞的凋亡率和蛋白印迹法(western blot)来观察细胞中特定蛋白质的变化.结果:①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功.②缺血/缺氧组与缺血/缺氧+磁场组比较,缺血缺氧组的凋亡率显著性增加,Akt磷酸化水平显著上升(P＜0.05).提示恒磁场可以使PI3K(Phosphoinositide-3kinase)/Akt(ProteinkinaseB,PKB)信号通路被激活而抑制凋亡的发生.结论:恒磁场通过激活PI3K/Akt信号通路抑制体外缺血缺氧条件下培养的骨髓间充质干细胞的凋亡.     

SDF-1α inhibits hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells through PI3K/Akt and ERK1/2 signaling pathways

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be a promising cell sources for cardiac regeneration. Poor survival rate of transplanted BMSCs in infarcted myocardium attenuated its clinical application. It’s reported that stromal-derived factor-1 (SDF-1) could protect progenitor cells including endothelial progenitor cells and embryonic stem cells from apoptosis. But little is known whether SDF-1α protein has the same protective effects on BMSCs under conditions of hypoxia and serum deprivation (hypoxia/SD). In present study, we verified that SDF-1α (0.50–2.0 μg/ml) inhibited hypoxia/SD induced apoptosis of BMSCs through mitochondrial pathway. After administration of SDF-1α, the loss of mitochondrial membrane potential and cytochrome c released from mitochondria to cytosol were significantly inhibited, and caspase 3 activity also declined. Furthermore, the effect of SDF-1α on mitochondrial pathway was neutralized by using PI3K inhibitor (Wortmannin) and ERK1/2 inhibitor (U0126). Our observations suggested that SDF-1α inhibits hypoxia/SD induced BMSCs apoptosis through PI3K/Akt and ERK1/2 signaling pathways. These data also imply that the anti-apoptotic effect mediated by SDF-1α may enhance cell survival after cell transplantation.     

Trichostatin A,an Inhibitor of Histone Deacetylase,Inhibits the Viability and Invasiveness of Hypoxic Rheumatoid Arthritis Fibroblast‐Like Synoviocytes via PI3K/Akt Signaling

This study was undertaken to explore the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, on the viability, apoptosis, and invasiveness of hypoxic rheumatoid arthritis fibroblast‐like synoviocytes (RA FLSs). RA FLSs were exposed to hypoxia for 24 h in the presence or absence of 2 μM TSA and tested for cell viability, apoptosis, invasion, and gene expression. The involvement of the phosphatidylinositol‐3‐kinase (PI3K)/Akt pathway was checked. TSA significantly inhibited the viability and induced apoptosis of hypoxic RA FLSs, compared to vehicle control. TSA blocked hypoxia‐induced invasion of RA FLSs during Matrigel invasion assays and reduced the expression of matrix metalloproteinases (MMP‐2 and MMP‐9) and PI3K and phosphorylation of Akt. Overexpression of constitutively active Akt reversed TSA‐mediated suppression of invasiveness and downregulation of MMP‐2 and MMP‐9. Our results indicate the antisurvival and antiinvasive activities of TSA in hypoxic RA FLSs, which is associated with inactivation of PI3K/Akt signaling.     

体外缺血缺氧条件下大鼠骨髓间充质干细胞凋亡发生的机制研究

目的：研究体外大鼠骨髓间充质干细胞（Bone marrow-derived mesenchymal stem cells, BMSCs）在缺血缺氧条件下发生凋亡的作用机制。方法：采取大鼠骨髓,以密度梯度离心分离出单个核细胞（MNCs）,于体外培养并由牛垂体提取物（PEX）诱导扩增传代培养出骨髓间充质干细胞（MSCs）。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,骨髓间充质干细胞（BMSCs）在缺血缺氧条件下培养,通过Annexin V／PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法（western blot）来观察细胞中蛋白的变化。结果：①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功。②对照组（无缺血缺氧）与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K（Phosphoinositide-3kinase）／Akt（ProteinkinaseB,PKB）信号通路被激活（P〈0．05）;同时缺血缺氧组与缺血缺氧＋PI3K／Akt抑制剂（LY294002）组比较,缺血缺氧＋PI3K／Akt抑制剂（LY294002）组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少提示PI3K/Akt信号通路被抑制（P〈0．05）。结论：PI3K／Akt信号通路对体外缺血缺氧条件下培养的骨髓间充质干细胞凋亡发生有关键性作用。     

Blocking exosomal miRNA-153-3p derived from bone marrow mesenchymal stem cells ameliorates hypoxia-induced myocardial and microvascular damage by targeting the ANGPT1-mediated VEGF/PI3k/Akt/eNOS pathway

It has been widely reported that exosomes derived from mesenchymal stem cells (MSCs) have a protective effect on myocardial infarction (MI). However, the specific molecules which play a damaging role in MSCs shuttled miRNAs are much less explored. MiRNA-153-3p (miR-153-3p) is a vital miRNA which has been proved to modulate cell proliferation, apoptosis, angiogenesis, peritoneal fibrosis and aortic calcification. Here, we aim to study the effect and mechanism of miR-153-3p in MSC-derived exosomes on hypoxia-induced myocardial and microvascular damage. The exosomes of MSCs were isolated and identified, and the MSCs-exosomes with low expression of miR-153-3p (exo-miR-153-3p⁻) were constructed to interfere with the endothelial cells and cardiomyocytes in the oxygen-glucose deprivation (OGD) model. The viability, apoptosis, angiogenesis of endothelial cells and cardiomyocytes were determined. Additionally, ANGPT1/VEGF/VEGFR2/PI3K/Akt/eNOS pathway was detected by ELISA and/or western blot. The results illustrated that exo-miR-153-3p⁻ significantly reduced the apoptosis of endothelial cells and cardiomyocytes and promoted their viability. Meanwhile, exo-miR-153-3p⁻ can promote the angiogenesis of endothelial cells. Mechanistically, miR-153-3p regulates the VEGF/VEGFR2/PI3K/Akt/eNOS pathways by targeting ANGPT1. Intervention with VEGFR2 inhibitor (SU1498, 1 μM) remarkably reversed the protective effect of exo-miR-153-3p⁻ in vascular endothelial cells and cardiomyocytes treated by OGD. Collectively, MSCs-derived exosomes with low-expressed miR-153-3p notably promotes the activation of ANGPT1 and the VEGF/VEGFR2 /PI3K/Akt/eNOS pathways, thereby preventing the damages endothelial cells and cardiomyocytes against hypoxia.     

The cardioprotective effect of dihydromyricetin prevents ischemia–reperfusion-induced apoptosis in vivo and in vitro via the PI3K/Akt and HIF-1α signaling pathways

Reperfusion therapy is widely used to treat acute myocardial infarction (AMI). However, further injury to the heart induced by rapidly initiating reperfusion is often encountered in clinical practice. A lack of pharmacological strategies in clinics limits the prognosis of patients with myocardial ischemia-reperfusion injury (MIRI). Dihydromyricetin (DMY) is one of the most abundant components in vine tea, commonly known as the tender stems and leaves of Ampelopsis grossedentata. The aim of this study was to evaluate the cardioprotection of DMY against myocardial ischemia-reperfusion (I/R) injury and to further investigate the underlying mechanism. An I/R injury was induced by left anterior descending coronary artery occlusion in adult male rats in vivo and a hypoxia–reoxygenation (H/R) injury in H9c2 cardiomyocytes in vitro. We found that DMY pretreatment provided significant protection against I/R-induced injury, including enhanced antioxidant capacity and inhibited apoptosis in vivo and in vitro. This effect correlated with the activation of the PI3K/Akt and HIF-1α signaling pathways. Conversely, blocking Akt activation with the PI3K inhibitor LY294002 effectively suppressed the protective effects of DMY against I/R-induced injury. In addition, the PI3K inhibitor partially blocked the effects of DMY on the upregulation of Bcl-2, Bcl-xl, procaspase-3, -8, and -9 protein expression and the downregulation of HIF-1α, Bnip3, Bax, Cyt-c, cleaved caspase-3, -8, and -9 protein expression. Collectively, these results showed that DMY decreased the apoptosis and necrosis by I/R treatment, and PI3K/Akt and HIF-1α plays a crucial role in protection during this process. These observations indicate that DMY has the potential to exert cardioprotective effects against I/R injury and the results might be important for the clinical efficacy of AMI treatment.     

U50,488H postconditioning reduces apoptosis after myocardial ischemia and reperfusion

AimsEvidence has indicated U50,488H, a selective κ-opioid receptor (κ-OR) agonist, administered before ischemia attenuates apoptosis and infarction during ischemia and reperfusion (I/R). However, it remains unclear whether U50,488H postconditioning reduces apoptosis during I/R. This study was designed, therefore, to test the hypothesis that U50,488H administered at the onset of reperfusion inhibits cardiomyocyte apoptosis and to investigate the underlying mechanisms.Main methodsMale Sprague–Dawley rats were subjected to myocardial ischemia and reperfusion(MI/R) and were randomized to receive either vehicle, U50,488H, U50,488H plus Nor-BNI, a selective κ-OR antagonist, U50,488H plus wortmannin, a specific inhibitor of phosphoinositide 3′-kinase (PI3K), or U50,488H plus L-NAME, a nitric oxide synthase inhibitor (NOS inhibitor), immediately prior to reperfusion. In vitro study was performed on cultured neonatal cardiomyocytes subjected to simulated ischemia/reperfusion.Key findingsTreatment with U50,488H resulted in increases in Akt and endothelial nitric oxide synthase (eNOS) phosphorylation with secondary NO production both in vivo and in vitro and these effect were completely blocked by wortmannin and specific Akt inhibitor(AI). L-NAME treatment had no effect on Akt and eNOS phosphorylation; but, significantly reduced NO production. Moreover, treatment with U50,488H markedly reduced myocardial apoptotic death. Treatment with wortmannin and specific Akt inhibitor abolished the anti-apoptotic effect of U50,488H. L-NAME also significantly attenuated the anti-apoptotic effect of U50,488H.SignificanceThese results demonstrate that U50,488H administered immediately prior to reperfusion increases Akt phosphorylation through a PI3-kinase-dependent mechanism and reduces postischemic myocardial apoptosis. Phosphorylation of eNOS with secondary NO production contribute significantly to the anti-apoptotic effect of U50,488H postconditioning.     

Transcriptional regulation of livin by β-catenin/TCF signaling in human lung cancer cell lines

Heregulin can regulate the survival of cardiomyocytes, epithelial cells, neuron, glial cells, and other cell types through binding with the ErbB receptors. The aim of this study is to investigate the effects of heregulin (HRG) on the apoptosis of Bone marrow Mesenchymal stem cells (MSCs). We used the MSCs from adult Sprague–Dawley rats and the model of serum deprivation (SD) and hypoxia-induced apoptosis. The apoptosis was detected by TUNEL method. The apoptosis of MSCs significantly increased 12 h or 18 h after SD and hypoxia, but treatment with HRG significantly decreased the apoptosis induced by SD and hypoxia. Tyrphostin AG1478 (ErbB3/4 inhibitor) or Tyrphostin AG825 (ErbB2 inhibitor) could block this effects of HRG. Akt and ERK were activated by HRG under SD and hypoxia conditions, but HRG had no effects on the activation of JNK and p38. HRG also increased the ratio of Bcl-2/Bax and decreased the activation of caspase3 induced by SD and hypoxia. These results suggested HRG could decrease the apoptosis of MSCs induced by SD and hypoxia through the activation of Akt and ERK, the increase of Bcl-2/Bax ratio and the inhibition of caspase3 activation.     

Differential role of PI3K/Akt pathway in the infarct size limitation and antiarrhythmic protection in the rat heart

Endogenous cardiac protection against prolonged ischemic insult can be achieved by repeated brief episodes of ischemia (hypoxia) or by cardiac adaptation to various stresses such as chronic hypoxia. Activation of phosphatidylinositol 3-kinase (PI3K)/Akt is involved in antiapoptotic effects, however, it is not clear whether it is required for overall heart salvage including protection against myocardial infarction and arrhythmias. We focussed on the potential common role of PI3K/Akt in anti-infarct protection, in the experimental settings of long-term adaptation to chronic intermittent hypobaric hypoxia (IHH; 8 h/day, 25–30 exposures, in vivo rats) and acute ischemic preconditioning (IP; Langendorff-perfused hearts). In addition, we explored the role of PI3K/Akt in susceptibility to ischemic ventricular arrhythmias. In normoxic open-chest rats, PI3K/Akt inhibitor LY294002 (LY; 0.3 mg/kg) given 5 min before test occlusion/reperfusion (I/R) did not affect infarct size (IS) normalized to the size of area at risk (AR). In hypoxic rats, LY partially attenuated IS-limiting effect of IHH (IS/AR 59.7 ± 4.1% vs. 51.8 ± 4.4% in the non-treated rats; p > 0.05) and increased IS/AR to its value in normoxic rats (64.9 ± 5.1%). In the isolated hearts, LY (5 μM) applied 15 min prior to I/R completely abolished anti-infarct protection by IP (IS/AR 55.0 ± 4.9% vs. 15.2 ± 1.2% in the non-treated hearts and 42.0 ± 5.5% in the non-preconditioned controls; p < 0.05). In the non-preconditioned hearts, PI3K/Akt inhibition did not modify IS/AR, on the other hand, it markedly suppressed arrhythmias. In the LY-treated isolated hearts, the total number of ventricular premature beats and the incidence of ventricular tachycardia (VT) was reduced from 518 ± 71 and 100% in the controls to 155 ± 15 and 12.5%, respectively (p < 0.05). Moreover, bracketing of IP with LY did not reverse antiarrhythmic effect of IP. These results suggest that activation of PI3K/Akt cascade plays a role in the IS-limiting mechanism in the rat heart, however, it is not involved in the mechanisms of antiarrhythmic protection.     

Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase 3 and apoptosis of cardiac muscle cells

Apoptosis of cardiac muscle cells contributes to the development of cardiomyopathy. Recent studies showed that insulin-like growth factor I (IGF-I) inhibits apoptosis of cardiac muscle cells and improves myocardial function in experimental heart failure. This study was carried out to elucidate the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the anti-apoptotic actions of IGF-I in cardiomyocytes and to explore whether expression of constitutively active PI 3-kinase can inhibit apoptosis in cardiomyocytes. Apoptosis of primary cardiomyocytes was induced by doxorubicin treatment and serum withdrawal. Transduction of cardiomyocytes with constitutively active PI 3-kinase specifically lead to serine phosphorylation of Akt, whereas phosphorylation of IGF-I receptor, IRS1/2 and p44/42 mitogen-activated protein kinase were not increased. In the cardiomyocytes transduced with constitutively active PI 3-kinase, activation of the pro-apoptotic caspase 3 was attenuated and fragmentation of DNA was reduced. Preincubating cells with PI 3-kinase inhibitor LY294002 was associated with loss of anti-apoptotic actions of IGF-I and PI 3-kinase. Neither IGF-I nor constitutively active PI 3-kinase lead to serine phosphorylation of Bad, suggesting that the anti-apoptotic effects of PI 3-kinase are not mediated through Bad phosphorylation in cardiac muscle cells. To determine whether activation of caspase 3 is sufficient to induce apoptosis in cardiomyocytes, an engineered TAT-caspase 3 protein was introduced to cardiomyocytes. Significant reduction of cell viability occurred in the cardiomyocytes transduced with active caspase 3, indicating that activation of caspase 3 is sufficient to cause cardiomyocyte death. These findings indicate the existence of an IGF-I receptor-PI 3-kinase-caspase 3 pathway in cardiomyocytes that plays an important role in the anti-apoptotic actions of IGF-I in heart. Moreover, these data suggest that modulation of PI 3-kinase activities may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in cardiomyopathy.     

A possible new activator of PI3K‐Huayu Qutan Recipe alleviates mitochondrial apoptosis in obesity rats with acute myocardial infarction

Obesity, which has unknown pathogenesis, can increase the frequency and seriousness of acute myocardial infarction (AMI). This study evaluated effect of Huayu Qutan Recipe (HQR) pretreatment on myocardial apoptosis induced by AMI by regulating mitochondrial function via PI3K/Akt/Bad pathway in rats with obesity. For in vivo experiments, 60 male rats were randomly divided into 6 groups: sham group, AMI group, AMI (obese) group, 4.5, 9.0 and 18.0 g/kg/d HQR groups. The models fed on HQR with different concentrations for 2 weeks before AMI. For in vitro experiments, the cardiomyocytes line (H9c2) was used. Cells were pretreated with palmitic acid (PA) for 24 h, then to build hypoxia model followed by HQR‐containing serum for 24 h. Related indicators were also detected. In vivo, HQR can lessen pathohistological damage and apoptosis after AMI. In addition, HQR improves blood fat levels, cardiac function, inflammatory factor, the balance of oxidation and antioxidation, as well as lessen infarction in rats with obesity after AMI. Meanwhile, HQR can diminish myocardial cell death by improving mitochondrial function via PI3K/Akt/Bad pathway activation. In vitro, HQR inhibited H9c2 cells apoptosis, improved mitochondrial function and activated the PI3K/Akt/Bad pathway, but effects can be peripeteiad by LY294002. Myocardial mitochondrial dysfunction occurs following AMI and can lead to myocardial apoptosis, which can be aggravated by obesity. HQR can relieve myocardial apoptosis by improving mitochondrial function via the PI3K/Akt/Bad pathway in rats with obesity.     

Hypoxia induces the activation of the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12 cells: protective role in apoptosis

Hypoxia is a common environmental stress that influences signaling pathways and cell function. Several cell types, including neuroendocrine chromaffin cells, have evolved to sense oxygen levels and initiate specific adaptive responses to hypoxia. Here we report that under hypoxic conditions, rat pheochromocytoma PC12 cells are resistant to apoptosis induced by serum withdrawal and chemotherapy treatment. This effect is also observed after treatment with deferoxamine, a compound that mimics many of the effects of hypoxia. The hypoxia-dependent protection from apoptosis correlates with activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is detected after 3-4 h of hypoxic or deferoxamine treatment and is sustained while hypoxic conditions are maintained. Hypoxia-induced Akt activation can be prevented by treatment with cycloheximide or actinomycin D, suggesting that de novo protein synthesis is required. Finally, inhibition of PI3K impairs both the protection against apoptosis and the activation of Akt in response to hypoxia, suggesting a functional link between these two phenomena. Thus, reduced oxygen tension regulates apoptosis in PC12 cells through activation of the PI3K/Akt survival pathway.     

Modulation of trophoblast cell death by oxygen and EGF

BACKGROUND: Preeclampsia, a maternal hypertensive disease, is characterized by shallow invasion of the maternal spiral arterioles resulting in hypoxia/reperfusion type insult; however, the molecular mechanism is unknown. The aim of this study was to determine the mechanism of altered oxygen tension or inhibition of phosphatidyl-inositol-3-kinase (PI3K) on trophoblast survival and to investigate the effect of epidermal growth factor (EGF) on maintaining cellular integrity. MATERIALS AND METHOD: We have used flow cytometry, immunoblotting, and fluoroimmunocytochemistry to study apoptosis in a characterized, spontaneously transformed first trimester extravillous-like trophoblast cell line that exhibits many characteristics of in vivo trophoblast. RESULTS: Time-dependent exposure of first trimester extravillous-like trophoblast to all oxygen tensions tested promoted dissipation of the mitochondrial membrane potential (psi(m)) and resulted in a significant increase in celldeath by 48 hr as determined by dual staining flow cytometry. Western blot analysis revealed expression ofcleaved caspase-3 and caspase-9 increased with time with hypoxia and hyperoxia promoting the greatest elevation indicating that longer duration of exposure to a change inoxygen tension causes increased apoptosis via a mitochondrial-mediated pathway. Disruption of the anti-apoptotic PI3K pathway by LY294002 (40 microM), its specific inhibitor, caused further significant dissipation of the psi(m) (p< 0.01) and cleavage of caspase-3. EGF was able to maintain the psi(m) and to prevent cleavage of caspase-3 even in the presence of LY294002, indicating that its survival effects were independent of the PI3K pathway. CONCLUSIONS: These results suggest that inhibition of the PI3K/Akt pathway can sensitize first-trimester trophoblast-like cells into oxygen-induced cell death and that EGF exerts its anti-apoptotic effect independently of PI3K/Akt.     

TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.Supported in part by the NIH grant PO1 CA55261     

Apelin-36, a potent peptide,protects against ischemic brain injury by activating the PI3K/Akt pathway

Apelin is an endogenous ligand of G protein-coupled receptor-apelin and angiotensin-1-like receptor (APJ). The biological effects of apelin–APJ system are reported in multiple systems including cardiovascular, endocrinal, and gastrointestinal system. Previous studies had shown that apelin-13 is a potential protective agent on cardiac ischemia; however, the role of apelin in the central nervous system remained unknown. In this study, we investigated therapeutic effects of apelin-36, a long form of apelin, in ischemic brain injury models. We found that apelin-36 reduced cerebral infarct volume in the middle cerebral artery occlusion (MCAO) model and the neonatal hypoxic/ischemic (H/I) injury model. Apelin-36 improved neurological deficits in the MCAO model and promoted long-term functional recovery after H/I brain injury. We further explored the protective mechanisms of apelin-36 on H/I brain injury. We clearly demonstrated that apelin-36 significantly reduced the levels of cleaved caspase-3 and Bax, two well-established apoptotic markers after H/I injury, indicating the anti-apoptotic activity of apelin-36 in ischemic injury. Since apelin-36 increased the level of phosphorylated Akt after H/I injury, we treated neonates with a specific PI3K inhibitor LY294002. We found that LY294002 decreased the phosphorylated Akt level and attenuated protective effects of apelin-36 on apoptosis. These suggested that the PI3K/Akt pathway was at least in part involved in the anti-apoptotic mechanisms of apelin-36. Our findings demonstrated that apelin-36 was a promising therapeutic agent on the treatment of ischemic brain injury.