Hypoxic Cell Death Research Articles

Synthesis of cytochrome c oxidase 2: a p53-dependent metabolic regulator that promotes respiratory function and protects glioma and colon cancer cells from hypoxia-induced cell death

P53 has an important role in the processing of starvation signals. P53-dependent molecular mediators of the Warburg effect reduce glucose consumption and promote mitochondrial function. We therefore hypothesized that the retention of wild-type p53 characteristic of primary glioblastomas limits metabolic demands induced by deregulated signal transduction in the presence of hypoxia and nutrient depletion. Here we report that short hairpin RNA-mediated gene suppression of wild-type p53 or ectopic expression of mutant temperature-sensitive dominant-negative p53(V135A) increased glucose consumption and lactate production, decreased oxygen consumption and enhanced hypoxia-induced cell death in p53 wild-type human glioblastoma cells. Similarly, genetic knockout of p53 in HCT116 colon carcinoma cells resulted in reduced respiration and hypersensitivity towards hypoxia-induced cell death. Further, wild-type p53 gene silencing reduced the expression of synthesis of cytochrome c oxidase 2 (SCO2), an effector necessary for respiratory chain function. An SCO2 transgene reverted the metabolic phenotype and restored resistance towards hypoxia in p53-depleted and p53 mutant glioma cells in a rotenone-sensitive manner, demonstrating that this effect was dependent on intact oxidative phosphorylation. Supplementation with methyl-pyruvate, a mitochondrial substrate, rescued p53 wild-type but not p53 mutant cells from hypoxic cell death, demonstrating a p53-mediated selective aptitude to metabolize mitochondrial substrates. Further, SCO2 gene silencing in p53 wild-type glioma cells sensitized these cells towards hypoxia. Finally, lentiviral gene suppression of SCO2 significantly enhanced tumor necrosis in a subcutaneous HCT116 xenograft tumor model, compatible with impaired energy metabolism in these cells. These findings demonstrate that glioma and colon cancer cells with p53 wild-type status can skew the Warburg effect and thereby reduce their vulnerability towards tumor hypoxia in an SCO2-dependent manner. Targeting SCO2 may therefore represent a valuable strategy to enhance sensitivity towards hypoxia and may complement strategies targeting glucose metabolism.

Transcriptional Targeting of Bone Morphogenetic Protein Receptors: Is it Ready for Prime Time?

Bone morphogenetic proteins (BMPs) have been shown to impact a variety of cellular functions, including apoptosis, hemostasis, proliferation, and cellular differentiation [ 1 Arihiro K. Inai K. Expression of CD31, Met/hepatocyte growth factor receptor and bone morphogenetic protein in bone metastasis of osteosarcoma. Pathol Int. 2001; 51: 100 Crossref PubMed Scopus (29) Google Scholar , 2 Raida M. Clement J.H. Ameri K. et al. Expression of bone morphogenetic protein 2 in breast cancer cells inhibits hypoxic cell death. Int J Oncol. 2005; 26: 1465 PubMed Google Scholar ]. Any discussion of the role of this complex set of proteins should begin with a brief overview of their function. BMPs are synthesized as 400–425 amino acid long precursor molecules that become mature after disulfide-linked dimerization with other BMP monomers [ 3 Sakou T. Bone morphogenetic proteins: From basic studies to clinical approaches. Bone. 1998; 22: 591 Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar ]. These active molecules then bind to one of four BMP type I (BMPR1) or two BMP type II (BMPR2) extracellular surface protein receptors [ 4 Kawabata M. Chytil A. Moses H.L. Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-β receptor. J Biol Chem. 1995; 270: 5625 Crossref PubMed Scopus (134) Google Scholar ]. BMP binding then facilitates the heteromeric association of BMPR1 and BMPR2 and subsequent phosphorylation of BMPR1 via the kinase activity of BMPR2 [ 5 Rosenzweig B.L. Imamura T. Okadome T. et al. Cloning and characterization of a human type II receptor for bone morphogenetic proteins. Proc Natl Acad Sci U S A. 1995; 92: 7632 Crossref PubMed Scopus (474) Google Scholar ]. SMAD proteins 1, 5, and 8 are then phosphorylated and dimerized with SMAD4 [ 6 Derynck R. Zhang Y. Feng X.H. Smads: Transcriptional activators of TGF-β responses. Cell. 1998; 95: 737 Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar , 7 Heldin C.H. Miyazono K. ten Dijke P. TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997; 390: 465 Crossref PubMed Scopus (3309) Google Scholar ]. This complex then translocates to the nucleus, where it triggers transcription of various gene targets. Because these molecules can serve as therapeutic targets for myriad diseases, they have been subjected to intense investigation over the past decade [ 8 Blish K.R. Wang W. Willingham M.C. et al. A human bone morphogenetic protein antagonist is down-regulated in renal cancer. Mol Biol Cell. 2008; 19: 457 Crossref PubMed Scopus (55) Google Scholar ]. Given the building interest in this area, one can reasonably wonder if research on molecular control of BMP is ready for primetime. The recent work by Dahdaleh et al. would suggest that BMP is a reasonable molecular target for the treatment of myriad diseases [ 9 Dahdaleh F.S. Carr J.C. Calva D. Howe J.R. Sp1 regulates the transcription of BMPR1A. J Surg Res. 2011; 171: e15 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar ].

Association of hypoxia inducible factor-1α polymorphisms with susceptibility to non–small-cell lung cancer

Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of cellular response to hypoxia and has been suggested to play an important role in tumorigenesis and metastasis. The aim of this study was to investigate the role of HIF-1α-1772 C/T (P582S) and -1790 G/A (A588T) polymorphisms in the susceptibility to and severity of non-small-cell lung cancer (NSCLC). Using a case-control study design and polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis, the allele frequencies and genotype distributions of each single nucleotide polymorphism in 285 NSCLC cases and 300 gender-matched controls were compared. The distribution of the genotype frequencies of HIF-1α-1772 C/T and -1790 G/A were significantly different between the NSCLC and the controls. Logistic regression analysis revealed that higher odds ratios (ORs) for lung cancer were observed for individuals with HIF-1α-1772 T/T genotype against CC/CT genotypes (an OR of 4.04, 95% confidence interval [CI] = 2.02-8.08, P = 0.0001), and HIF-1α-1790 A/A genotype against GG/GA genotypes (an OR of 4.42, 95% CI 2.22-8.78, P < 0.0001). There were no relationship between HIF-1α-1772 C/T or -1790 G/A allele distribution and disease severity of NSCLC (P > 0.05). However, those patients carrying a HIF-1α-1772 T/T genotype or a HIF-1α-1790 A/A had a tendency toward inferior prognosis compared with other patients.

Growth of necrotic cores in atherosclerotic plaque

Plaques are fatty deposits that grow mainly in arteries and develop as a result of a chronic inflammatory response. Plaques are characterized as 'vulnerable' when they have large internal regions of necrosis and are heavily infiltrated by macrophages. The particular composition of a vulnerable plaque renders it susceptible to rupture, which releases thrombogenic agents into the bloodstream and can result in myocardial infarction. In this paper, we propose a mathematical model to predict the development of a plaque's necrotic core. By solving coupled reaction-diffusion equations for macrophages and dead cells, we focus on the joint effects of hypoxic cell death and chemoattraction to oxidized low-density lipoprotein (Ox-LDL), a molecule that is strongly linked to atherosclerosis. We do not model the mechanical properties of the plaque, its growth or rupture. Our model predicts cores that have approximately the right size and shape when compared to ultrasound images. Because our model is linear and autonomous, normal mode analysis and subsequent calculation of the smallest eigenvalue allow us to compute the times taken for the necrotic core to form. We find that the spatial distribution of Ox-LDL within the plaque determines not only the placement and size of cores, but their time of formation. Although plaques are biochemically complex, our study shows that certain aspects of their composition can be predicted and are, in fact, governed by simple physical models.

HIF-1α in Epidermis: Oxygen Sensing, Cutaneous Angiogenesis, Cancer, and Non-Cancer Disorders

Besides lung, postnatal human epidermis is the only epithelium in direct contact with atmospheric oxygen. Skin epidermal oxygenation occurs mostly through atmospheric oxygen rather than tissue vasculature, resulting in a mildly hypoxic microenvironment that favors increased expression of hypoxia-inducible factor-1α (HIF-1α). Considering the wide spectrum of biological processes, such as angiogenesis, inflammation, bioenergetics, proliferation, motility, and apoptosis, that are regulated by this transcription factor, its high expression level in the epidermis might be important to HIF-1α in skin physiology and pathophysiology. Here, we review the role of HIF-1α in cutaneous angiogenesis, skin tumorigenesis, and several skin disorders.

Overcoming Hypoxia-Induced Apoptotic Resistance through Combinatorial Inhibition of GSK-3β and CDK1

Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we conducted a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds, we identified were a structurally related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3β and cyclin-dependent kinase (CDK) 1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator hypoxia-inducible factor (HIF) 1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3β and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.

Nuclear factor-κB2/p100 promotes endometrial carcinoma cell survival under hypoxia in a HIF-1α independent manner

Endometrial carcinoma (EC) is a common female cancer, treated mainly by surgery and adjuvant radiotherapy. Relapse following treatment is associated with increased risk of metastases. Hypoxia, a common microenvironment in solid tumors, correlates with malignant progression, rendering tumors resistant to ionizing therapy. Hence, we assessed here the immunohistochemical expression of hypoxia-inducible factor-1α (HIF-1α) and members of the NF-κB family in 82 primary EC and 10 post-radiation recurrences of EC. Post-radiation recurrences were highly hypoxic, with a higher expression of HIF-1α and also RelA (p65) and p52 when compared with primary EC. We next investigated the effects of hypoxia on EC cell lines. We found that EC cell lines are highly resistant to hypoxia-induced apoptosis. We thus focused on the molecular mechanisms involved in conferring hypoxic cell death resistance. We show that in addition to the classical NF-κB, hypoxia activates the alternative NF-κB pathway. To characterize the upstream kinases involved in the activation of these pathways, we used lentiviral-mediated knockdown and mouse embryonic fibroblasts lacking IKKα and IKKβ kinases. Both IKKα and IKKβ kinases are required for RelA (p65) and p100 accumulation, whereas p52 processing under hypoxia is IKKα dependent. Furthermore, Ishikawa endometrial cell line harboring either RelA (p65) or p52 short-hairpin RNA was sensitive to hypoxia-induced cell death, indicating that, in addition to the known prosurvival role of RelA (p65) under hypoxia, alternative NF-κB pathway also enhances hypoxic survival of EC cells. Interestingly, although HIF-1α controlled classical NF-κB activation pathway and survival under hypoxia through RelA (p65) nuclear accumulation, the alternative pathway was HIF-1α independent. These findings have important clinical implications for the improvement of EC prognosis before radiotherapy.

Aldehyde dehydrogenase 2 (ALDH2) rescues myocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic aldehyde

The present study was designed to examine the mechanism involved in mitochondrial aldehyde dehydrogenase (ALDH2)-induced cardioprotection against ischaemia/reperfusion (I/R) injury with a focus on autophagy. Wild-type (WT), ALDH2 overexpression, and knockout (KO) mice (n = 4-6 for each index measured) were subjected to I/R, and myocardial function was assessed using echocardiographic, Langendroff, and edge-detection systems. Western blotting was used to evaluate AMP-dependent protein kinase (AMPK), Akt, autophagy, and the AMPK/Akt upstream signalling LKB1 and PTEN. ALDH2 overexpression and KO significantly attenuated and accentuated, respectively, infarct size, factional shortening, and recovery of post-ischaemic left ventricular function following I/R as well as hypoxia/reoxygenation-induced cardiomyocyte contractile dysfunction. Autophagy was induced during ischaemia and remained elevated during reperfusion. ALDH2 significantly promoted autophagy during ischaemia, which was accompanied by AMPK activation and mammalian target of rapamycin (mTOR) inhibition. On the contrary, ALDH2 overtly inhibited autophagy during reperfusion accompanied by the activation of Akt and mTOR. Inhibition and induction of autophagy mitigated ALDH2-induced protection against cell death in hypoxia and reoxygenation, respectively. In addition, levels of the endogenous toxic aldehyde 4-hydroxy-2-nonenal (4-HNE) were elevated by ischaemia and reperfusion, which was abrogated by ALDH2. Furthermore, ALDH2 ablated 4-HNE-induced cardiomyocyte dysfunction and protein damage, whereas 4-HNE directly decreased pan and phosphorylated LKB1 and PTEN expression. Our data suggest a myocardial protective effect of ALDH2 against I/R injury possibly through detoxification of toxic aldehyde and a differential regulation of autophagy through AMPK- and Akt-mTOR signalling during ischaemia and reperfusion, respectively.

Effect of mild and moderate hypothermia on hypoxic injury in nearly pure neuronal culture

The effects of mild and moderate hypothermic therapy on cerebral injury are still controversial. Our hypothesis is that mild and moderate hypothermia should have some effects on neurons themselves if they really have protective effects. By using a nearly pure neuronal culture, we evaluated the effects and mechanism of hypothermia against hypoxic insult. A nearly pure neuronal culture from cortices of 18-day-old Wister rats was used. The neurons were exposed to below 1% oxygen at 3 different temperatures (30, 33 and 37°C). First, cell viability was measured by assessing viable neurons with trypan blue. Second, to evaluate the mechanism, the extracellular glutamate concentration was measured by high-performance liquid chromatography after hypoxia; cell viability after exposure to extrinsic glutamate was also evaluated. Next, mitochondrial membrane potential was estimated, by monitoring aggregation of MitoCapture™, and the percentage of apoptotic cells was evaluated by staining with Hoechst 33342 and propidium iodide. After 24-h hypoxic insult, cell viability at 30 and 33°C was significantly higher than at 37°C. There was no significant difference between extracellular concentrations of glutamate after hypoxia or cell viability after glutamate exposure among the 3temperature groups. In moderate hypothermia, the number of neurons with mitochondrial injury and the percentage of apoptotic cells were significantly reduced. Mild and moderate hypothermia inhibited hypoxic neuronal cell death. The mechanism of this effect may be related to protection of mitochondrial function, presumably followed by inhibition of apoptosis, at least in moderate hypothermia.

Human PRP19 interacts with prolyl-hydroxylase PHD3 and inhibits cell death in hypoxia

Prolyl-hydroxylase PHDs are the key regulators of hypoxia-inducible factor (HIF) stability. PHD3 has been shown to form a large complex under hypoxic conditions. While attempting to characterize the complex by determining its components, we identified human PRP19. hPRP19 is a multi-functional protein that plays a role in splicing, ubiquitination, and cell growth. Here, we report that PHD3 efficiently forms a complex with hPRP19 under hypoxic conditions and prevents cell death under prolonged hypoxic conditions. hPRP19 interacts with PHD3 via its C-terminal WD40 region, and the interaction is enhanced under hypoxic conditions through the utilization of the N-terminal coiled-coil domain. Cell death observed under prolonged hypoxic conditions is suppressed by the forced expression of hPRP19 in PC12 and HEK293T cells. In contrast, hPRP19 silencing by siRNA increased the caspase activity and enhanced cell death under hypoxic conditions in HeLa cells. Further, silencing of both PHD3 and hPRP19 recovers the cell death induced by hPRP19 single siRNA. Taken together, the results of our study indicate that hPRP19 interacts with PHD3 to suppress the cell death under hypoxic conditions by limiting the function of PHD3 which leads to caspase activation.

Bifurcate effects of glucose on caspase-independent cell death during hypoxia

We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.

Abstract 87: Differential effects of JNK1 and JNK2 on cell death in hypoxic colon cancer cell lines

Abstract BACKGROUND: We previously showed that hypoxic colon cancer cell lines exhibit higher sensitivity to oxaliplatin when SEK1 function is impaired, and greater resistance when MKK7 is down regulated. The MKK7 mutant lines exhibit more profound inhibition of JNK activation than those lacking SEK1, and their effects on the activation of JNK1 and JNK2 differ. The HT29 cells do not die readily by apoptosis under these conditions, and both oxaliplatin and hypoxia induce autophagy in these cells. PURPOSE: The purpose of this study is to investigate further how modulation of signaling through JNK1 and JNK2 influences cell death pathways in hypoxic HT29 cells treated with oxaliplatin. For that, we employed HT29-derived cell lines stably expressing empty vector (HT.LX) and dominant negative constructs for JNK1 (HTJ1.3) and JNK2 (HTJ2.2). RESULTS: We observed higher induction and activation of c-Jun in cells with dnJNK1, while introduction of dnJNK2 caused the opposite effect. Resistance to oxaliplatin under hypoxic conditions was also higher in the cell line with lowest c-Jun activation level (IC50 of 2.1 μM for HT.LX, 3 μM for HTJ2.2 and 0.8 μM for HTJ1.3), suggesting a pro-survival role for JNK1 under these conditions. Both oxaliplatin- and hypoxia-induced formation of autophagosomes was reduced significantly in HT29 cells treated with the JNK inhibitor SP600125. We also found differential sensitivity of these cell lines to an inhibitor of autophagy, chloroquine (CQ): HTJ1.3 and HTJ2.2 cell lines were slightly more resistant to CQ under normal conditions, as compared to parental line, but under hypoxia all of the lines demonstrated higher sensitivity to the drug. Notably, in light of the role of JNK1 in activating autophagy, cells with diminished JNK1 activity were the most sensitive to CQ under hypoxia. Moreover, when we treated the cell lines with 5 μM of CQ under hypoxia/reoxygenation, inhibition of autophagy and induction of apoptosis were most significant in dnJNK1-expressing cells. This effect of JNK1 down-regulation is specific to hypoxia, since the constructs do not differ in sensitivity to CQ and oxaliplatin in combination. CONCLUSIONS: Our data demonstrate the differential effects of JNK1/JNK2 modulation on cell death of hypoxic HT29 cells treated with oxaliplatin or chloroquine, and imply pro-survival function for JNK1 versus JNK2 under these conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 87.

Calcium fluxes cause nuclear shrinkage and the translocation of phospholipase C-δ1 into the nucleus

Phospholipase C-δ1 (PLCδ1) is the most fundamental form of the eukaryotic PLC and thought to play important roles in the regulation of cells. We previously reported that PLCδ1 shuttles between the cytoplasm and nucleus, and an influx of Ca 2+ triggers the nuclear import of PLCδ1 via Ca 2+-dependent interaction with importin β1, although the physiological meaning of this is unclear. Here we have examined the distribution of PLCδ1 using primary cultures of rat hippocampal neurons. Treatment of 7DIV neurons with ionomycin or thapsigargin caused the nuclear localization of PLCδ1 as has been observed in other cell lines. Similar results were obtained with neurons treated with glutamate, suggesting that the nuclear localization of PLCδ1 plays some roles in excitotoxicity associated with ischemic stress. Generally, cells undergoing ischemic or hypoxic cell death show nuclear shrinkage. We confirmed that a massive influx of Ca 2+ caused similar results. Furthermore, overexpression of GFP-PLCδ1 facilitated ionomycin-induced nuclear shrinkage in embryonic fibroblasts derived from PLCδ1 gene-knockout mice (PLCδ1KO-MEF). By contrast, an E341A mutant that cannot bind with importin β1 and be imported into the nucleus by ionomycin and also lacks enzymatic activity did not cause nuclear shrinkage in PLCδ1KO-MEF. Nuclear translocation and the PLC activity of PLCδ1, therefore, may regulate the nuclear shape by controlling the nuclear scaffold during stress-induced cell death caused by high levels of Ca 2+.

Hypoxia-induced neuronal apoptosis is mediated by de novo synthesis of ceramide through activation of serine palmitoyltransferase

Cellular hypoxia can lead to cell death or adaptation and has important effects on development, physiology, and pathology. Here, we investigated the role and regulation of ceramide in hypoxia-induced apoptosis of SH-SY5Y neuroblastoma cells. Hypoxia increased the ceramide concentration; subsequently, we observed biochemical changes indicative of apoptosis, such as DNA fragmentation, nuclear staining, and poly ADP-ribose polymerase (PARP) cleavage. The hypoxic cell death was potently inhibited by a caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone). l-Cycloserine, a serine palmitoyltransferase (SPT) inhibitor, and fumonisin B 1 (FB 1), a ceramide synthase inhibitor, inhibited the hypoxia-induced increase in ceramide, indicating that the increase occurred via the de novo pathway. Hypoxia increased the activity and protein levels of SPT2, suggesting that the hypoxia-induced increase in ceramide is due to the transcriptional up-regulation of SPT2. Specific siRNA of SPT2 prevented hypoxia-induced cell death and ceramide production. However, hypoxia also increased the cellular level of glucosylceramide, which was inhibited by a glucosylceramide synthase (GCS) inhibitor and specific siRNA, but not a ceramidase inhibitor. The increase in glucosylceramide was accompanied by increases in both PARP cleavage and DNA fragmentation. Together, the current results suggest that both SPT and GCS may regulate the cellular level of ceramide, and thus may be critical enzymes for deciding the fate of the cells exposed to hypoxia.

Adrenomedullin protects against hypoxia/reoxygenation-induced cell death by suppression of reactive oxygen species via thiol redox systems

Adrenomedullin (ADM) functions as a survival factor against hypoxic cell death. However, molecular mechanisms underlying the cell survival pathway remain largely unknown. In this report, we showed that ADM suppressed reactive oxygen species (ROS) increase by inhibiting reduction of glutathione (GSH) level in hypoxia/reoxygenation (H/R) injury, and increased the activities of glutathione peroxidase and reductase. In addition, ADM maintained total and active reduced thioredoxin (Trx) levels against H/R. We also found that ADM blocked nuclear translocation of Trx induced by H/R. The results of the present study show that ADM regulates cellular ROS levels via the GSH and Trx system.

STIM2 Regulates Capacitive Ca 2+ Entry in Neurons and Plays a Key Role in Hypoxic Neuronal Cell Death

Excessive cytosolic calcium ion (Ca(2+)) accumulation during cerebral ischemia triggers neuronal cell death, but the underlying mechanisms are poorly understood. Capacitive Ca(2+) entry (CCE) is a process whereby depletion of intracellular Ca(2+) stores causes the activation of plasma membrane Ca(2+) channels. In nonexcitable cells, CCE is controlled by the endoplasmic reticulum (ER)-resident Ca(2+) sensor STIM1, whereas the closely related protein STIM2 has been proposed to regulate basal cytosolic and ER Ca(2+) concentrations and make only a minor contribution to CCE. Here, we show that STIM2, but not STIM1, is essential for CCE and ischemia-induced cytosolic Ca(2+) accumulation in neurons. Neurons from Stim2(-/-) mice showed significantly increased survival under hypoxic conditions compared to neurons from wild-type controls both in culture and in acute hippocampal slice preparations. In vivo, Stim2(-/-) mice were markedly protected from neurological damage in a model of focal cerebral ischemia. These results implicate CCE in ischemic neuronal cell death and establish STIM2 as a critical mediator of this process.

Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death

Cells exposed to low-oxygen conditions (hypoxia) alter their metabolism to survive. This response, although vital during development and high-altitude survival, is now known to be a major factor in the selection of cells with a transformed metabolic phenotype during tumorigenesis. It is thought that hypoxia-selected cells have increased invasive capacity and resistance to both chemo- and radiotherapies, and therefore represent an attractive target for antitumor therapy. Hypoxia inducible factors (HIFs) are responsible for the majority of gene expression changes under hypoxia, and are themselves controlled by the oxygen-sensing HIF prolyl hydroxylases (PHDs). It was previously shown that mutations in succinate dehydrogenase lead to the inactivation PHDs under normoxic conditions, which can be overcome by treatment with alpha-ketoglutarate derivatives. Given that solid tumors contain large regions of hypoxia, the reactivation of PHDs in these conditions could induce metabolic catastrophe and therefore prove an effective antitumor therapy. In this report we demonstrate that derivatized alpha-ketoglutarate can be used as a strategy for maintaining PHD activity under hypoxia. By increasing intracellular alpha-ketoglutarate and activating PHDs we trigger PHD-dependent reversal of HIF1 activation, and PHD-dependent hypoxic cell death. We also show that derivatized alpha-ketoglutarate can permeate multiple layers of cells, reducing HIF1alpha levels and its target genes in vivo.

Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid‐based implantation model

Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co-implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular-derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC). EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice. EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE-cadherin and vascular endothelial growth factor-R2, and in their ability to endocytose acetylated low-density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro, as assessed by in vitro Matrigel sprouting assay. However in vivo, a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions. Our results indicate that vascular-derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.

Insufficiency of Pro-heparin-binding Epidermal Growth Factor-like Growth Factor Shedding Enhances Hypoxic Cell Death in H9c2 Cardiomyoblasts via the Activation of Caspase-3 and c-Jun N-terminal Kinase

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cardiogenic and cardiohypertrophic growth factor. ProHB-EGF, a product of the Hb-egf gene and the precursor of HB-EGF, is anchored to the plasma membrane. Its ectodomain region is shed by a disintegrin and metalloproteases (ADAMs) when activated by various stimulations. It has been reported that an uncleavable mutant of Hb-egf, uc-Hb-egf, produces uc-proHB-EGF, which is not cleaved by ADAMs and causes dilation of the heart in knock-in mice. This suggests that the shedding of proHB-EGF is essential for the development and survival of cardiomyocytes: however, the molecular mechanism involved has remained unclear. In this study, we investigated the relationship between uc-proHB-EGF expression and cardiomyocyte survival. Human uc-proHB-EGF was adenovirally introduced into the rat cardiomyoblast cell line H9c2, and the cells were cultured under normoxic and hypoxic conditions. Uc-proHB-EGF-expressing H9c2 cells underwent apoptosis under normoxic conditions, which distinctly increased under hypoxic conditions. Furthermore, we observed an increased Caspase-3 activity, reactive oxygen species accumulation, and an increased c-Jun N-terminal kinase (JNK) activity in the uc-proHB-EGF-expressing H9c2 cells. Treatment of the uc-proHB-EGF transfectants with inhibitors of Caspase-3, reactive oxygen species, and JNK, namely, Z-VAD-fmk, N-acetylcysteine, and SP600125, respectively, significantly reduced hypoxic cell death. These data indicate that insufficiency of proHB-EGF shedding under hypoxic stress leads to cardiomyocyte apoptosis via Caspase-3- and JNK-dependent pathways.

The polyamines regulate endothelial cell survival during hypoxic stress through PI3K/AKT and MCL-1

Hypoxia-dependent angiogenesis is an inherent feature of solid tumors, and a better understanding of the molecular mechanisms of hypoxic cell-death should provide additional targets for cancer therapy. Here, we show a novel role of the polyamines in endothelial cell (EC) survival during hypoxia. Polyamine depletion by specific inhibition of ornithine decarboxylase was shown to protect ECs from hypoxia-induced apoptosis. Inhibition of the polyamines resulted in a significant induction of PI3K/AKT and its down-stream target MCL-1, i.e. an anti-apoptotic member of the BCL-2 family. Specific inhibitors of PI3K reversed the decrease of hypoxia-induced apoptosis as well as the induction of MCL-1 in polyamine-deprived cells. Moreover, siRNA-mediated down-regulation of MCL-1 was found to counter-act the protective effect of polyamine inhibition. We conclude that the polyamines regulate hypoxia-induced apoptosis in ECs through PI3K/AKT and MCL-1 dependent pathways. Our results may have important implications for the modulation of hypoxia-driven neovascularization.