Survivin-Dependent Angiogenesis in Ischemic Brain
2003; Elsevier BV; Volume: 163; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63453-0
ISSN1525-2191
AutoresEdward M. Conway, Femke Zwerts, Veerle Van Eygen, Astrid DeVriese, Nobuo Nagai, Wei Luo, Désiré Collen,
Tópico(s)MicroRNA in disease regulation
ResumoApproaches to regulating angiogenesis in the brain, which may diminish parenchymal damage after stroke, are lacking. Survivin, the inhibitor of apoptosis protein, is up-regulated in vitro in vascular endothelial cells by angiogenic factors, including vascular endothelial cell growth factor (VEGF). To evaluate the in vivo role of survivin in the brain in response to hypoxia/ischemia, we used a mouse model of stroke and show that 2 days after permanent middle cerebral artery occlusion, survivin is uniquely expressed by microvessels that form in the peri-infarct and infarct regions. The extent of vascularization of the infarct is dependent on expression of survivin, since vessel density is significantly reduced in mice with heterozygous deficiency of the survivin gene (survivin+/− mice), even though infarct sizes were not different. Hypoxia alone induces survivin expression in the brain, by cultured endothelial cells and by embryonic stem cells, but this response is at least partially independent of VEGF, hypoxia inducible factor 1α, or placental growth factor. Delineating the spatiotemporal pattern of expression of survivin after stroke, and the molecular mechanisms by which this is regulated, may provide novel approaches to therapeutically optimize angiogenesis in a variety of ischemic disorders. Approaches to regulating angiogenesis in the brain, which may diminish parenchymal damage after stroke, are lacking. Survivin, the inhibitor of apoptosis protein, is up-regulated in vitro in vascular endothelial cells by angiogenic factors, including vascular endothelial cell growth factor (VEGF). To evaluate the in vivo role of survivin in the brain in response to hypoxia/ischemia, we used a mouse model of stroke and show that 2 days after permanent middle cerebral artery occlusion, survivin is uniquely expressed by microvessels that form in the peri-infarct and infarct regions. The extent of vascularization of the infarct is dependent on expression of survivin, since vessel density is significantly reduced in mice with heterozygous deficiency of the survivin gene (survivin+/− mice), even though infarct sizes were not different. Hypoxia alone induces survivin expression in the brain, by cultured endothelial cells and by embryonic stem cells, but this response is at least partially independent of VEGF, hypoxia inducible factor 1α, or placental growth factor. Delineating the spatiotemporal pattern of expression of survivin after stroke, and the molecular mechanisms by which this is regulated, may provide novel approaches to therapeutically optimize angiogenesis in a variety of ischemic disorders. Following acute cerebral ischemia due to diminished blood supply to the brain, deprivation of oxygen and glucose results in a series of biochemical events, leading eventually to cell death and often devastating functional neurological disturbances. Observations that postischemic neovascularization in the infarct and peri-infarct regions correlated with survival in patients with stroke1Krupinski J Kaluza J Kumar P Kumar S Wang JM Role of angiogenesis in patients with cerebral ischemic stroke.Stroke. 1994; 25: 1794-1798Crossref PubMed Scopus (711) Google Scholar strongly suggested that angiogenesis might be a compensatory protective mechanism, and thus a potential therapeutic target. Therefore, major efforts are being made to delineate the molecular events that regulate angiogenesis in the setting of stroke. In rodent stroke models, neovascularization is evident within 1 to 3 days after middle cerebral artery occlusion (MCAO).2Marti HJ Bernaudin M Bellail A Schoch H Euler M Petit E Risau W Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia.Am J Pathol. 2000; 156: 965-976Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar This occurs concomitantly with increased expression, by neurons, microglial cells, astrocytes and vessels, of the angiogenic molecule, vascular endothelial growth factor (VEGF).3Plate KH Mechanisms of angiogenesis in the brain.J Neuropathol Exp Neurol. 1999; 58: 313-320Crossref PubMed Scopus (308) Google Scholar, 4Beck H Acker T Puschel AW Fujisawa H Carmeliet P Plate KH Cell type-specific expression of neuropilins in an MCA-occlusion model in mice suggests a potential role in post-ischemic brain remodeling.J Neuropathol Exp Neurol. 2002; 61: 339-350Crossref PubMed Scopus (95) Google Scholar VEGF is the most prominent of the angiogenic factors, exerting its effects predominantly via VEGF tyrosine kinase receptors, VEGFR-1 and VEGFR-2, and neuropilins-1 and -2 (NP-1 and NP-2) (for review5Carmeliet P Storkebaum E Vascular and neuronal effects of VEGF in the nervous system: implications for neurological disorders.Semin Cell Dev Biol. 2002; 13: 39-53Crossref PubMed Scopus (214) Google Scholar). VEGF augments vascular endothelial cell growth and migration, but is also a potent vascular permeability factor. It further interferes with vascular endothelial apoptosis by promoting expression of inhibitors of apoptosis, including survivin6Tran J Rak J Sheehan C Saibil SD LaCasse E Korneluk RG Kerbel RS Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells.Biochem Biophys Res Commun. 1999; 264: 781-788Crossref PubMed Scopus (308) Google Scholar, 7O'Connor DS Schechner JS Adida C Mesri M Rothermel AL Li F Nath AK Pober JS Altieri DC Control of apoptosis during angiogenesis by survivin expression in endothelial cells.Am J Pathol. 2000; 156: 393-398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar and other cell-survival proteins such as Akt.8Zhu WH MacIntyre A Nicosia RF Regulation of angiogenesis by vascular endothelial growth factor and angiopoietin-1 in the rat aorta model: distinct temporal patterns of intracellular signaling correlate with induction of angiogenic sprouting.Am J Pathol. 2002; 161: 823-830Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar While minimally expressed in normal adult brain,9Croll SD Wiegand SJ Vascular growth factors in cerebral ischemia.Mol Neurobiol. 2001; 23: 121-135Crossref PubMed Scopus (75) Google Scholar VEGF rapidly accumulates after ischemia, appearing in the peri-infarct and infarct regions10Lennmyr F Ata KA Funa K Olsson Y Terent A Expression of vascular endothelial growth factor (VEGF) and its receptors (Flt-1 and Flk-1) following permanent and transient occlusion of the middle cerebral artery in the rat.J Neuropathol Exp Neurol. 1998; 57: 874-882Crossref PubMed Scopus (234) Google Scholar and persisting for at least 7 days.11Plate K Beck H Danner S Allegrini P Wiessner C Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct.J Neuropath Exper Neurol. 1999; 58: 654-666Crossref PubMed Scopus (223) Google Scholar The postischemia neovascular response, driven largely by hypoxia-induced up-regulation and stabilization of VEGF, is initiated and maintained at least in part via hypoxia-inducible transcription factors, HIF1α and HIF2α.2 In concert with enhanced expression of VEGF and its receptors, other growth factors and cellular receptors, including placental growth factor (Plgf), angiopoietin-1 and angiopoietin-2, tie-1 and tie-2,12Lin TN Wang CK Cheung WM Hsu CY Induction of angiopoietin and Tie receptor mRNA expression after cerebral ischemia-reperfusion.J Cereb Blood Flow Metab. 2000; 20: 387-395Crossref PubMed Scopus (131) Google Scholar, 13Beck H Acker T Wiessner C Allegrini PR Plate KH Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat.Am J Pathol. 2000; 157: 1473-1483Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar basic fibroblast growth factor (bFGF), thrombospondin-1 and thrombospondin-2,14Lin TN Kim GM Chen JJ Cheung WM He YY Hsu CY Differential regulation of thrombospondin-1 and thrombospondin-2 after focal cerebral ischemia/reperfusion.Stroke. 2003; 34: 177-186Crossref PubMed Scopus (138) Google Scholar are also differentially regulated in highly specific spatio-temporal expression patterns, orchestrated to acutely minimize parenchymal damage, and to optimize subsequent healing and recovery (for review see9Croll SD Wiegand SJ Vascular growth factors in cerebral ischemia.Mol Neurobiol. 2001; 23: 121-135Crossref PubMed Scopus (75) Google Scholar, 15Zhang Z Chopp M Vascular endothelial growth factor and angiopoietins in focal cerebral ischemia.Trends Cardiovasc Med. 2002; 12: 62-66Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Armed with these new insights, angiogenic agents are currently being evaluated for treating stroke. In this regard, a major focus has been directed toward testing the efficacy of VEGF. Administration of VEGF to rats 48 hours after middle cerebral artery (MCA) ischemia yielded beneficial effects with improved neurological recovery associated with enhanced angiogenesis.16Zhang ZG Zhang L Jiang Q Zhang R Davies K Powers C Bruggen N Chopp M VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain.J Clin Invest. 2000; 106: 829-838Crossref PubMed Scopus (1072) Google Scholar Furthermore, in a transient model of MCAO in rats, continuous infusion of VEGF into the lateral ventricle reduced infarct volume and cerebral edema, and appeared to have a direct neuroprotective effect.17Harrigan MR Ennis SR Sullivan SE Keep RF Effects of intraventricular infusion of vascular endothelial growth factor on cerebral blood flow, edema, and infarct volume.Acta Neurochir (Wien). 2003; 145: 49-53Crossref PubMed Scopus (89) Google Scholar Nitric oxide, administered 24 hours after cerebral artery occlusion in rats, also enhanced angiogenesis in the ischemic brain, a response that was in part mediated by VEGF.18Zhang R Wang L Zhang L Chen J Zhu Z Zhang Z Chopp M Nitric oxide enhances angiogenesis via the synthesis of vascular endothelial growth factor and cGMP after stroke in the rat.Circ Res. 2003; 92: 308-313Crossref PubMed Scopus (256) Google Scholar In contrast to these promising findings, however, administration of VEGF after 1 hour of ischemia was complicated by blood brain barrier (BBB) leakage, hemorrhage, and larger ischemic lesions.16Zhang ZG Zhang L Jiang Q Zhang R Davies K Powers C Bruggen N Chopp M VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain.J Clin Invest. 2000; 106: 829-838Crossref PubMed Scopus (1072) Google Scholar This undesirable outcome, believed to result from vascular permeability-inducing properties of VEGF, was not observed when VEGF was administered directly and somewhat later to the surface of the postischemic brain.19Hayashi T Abe K Itoyama Y Reduction of ischemic damage by application of vascular endothelial growth factor in rat brain after transient ischemia.J Cereb Blood Flow Metab. 1998; 18: 887-895Crossref PubMed Scopus (301) Google Scholar The early injurious effect of VEGF likely reflected relatively low-level expression of vasculo-protective molecules, such as angiopoietin-1, that at later postischemia time points are increased and protect the stability of the vessels.9Croll SD Wiegand SJ Vascular growth factors in cerebral ischemia.Mol Neurobiol. 2001; 23: 121-135Crossref PubMed Scopus (75) Google Scholar, 13Beck H Acker T Wiessner C Allegrini PR Plate KH Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat.Am J Pathol. 2000; 157: 1473-1483Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar Thus, administration of angiopoietin-1, believed to enhance vascular integrity, plus VEGF, provided some protection against VEGF-induced BBB leakage shortly after focal ischemia.20Zhang ZG Zhang L Croll SD Chopp M Angiopoietin-1 reduces cerebral blood vessel leakage and ischemic lesion volume after focal cerebral embolic ischemia in mice.Neuroscience. 2002; 113: 683-687Crossref PubMed Scopus (135) Google Scholar Despite these advances, major gains in terms of stroke management have yet to be seen in the clinic. Further research is required to delineate the molecular mechanisms by which angiogenesis proceeds in the postischemic brain, so that a stable vascular network can be rapidly provided to rescue neurons from apoptosis/necrosis and to promote healing. As noted, VEGF also promotes vascular endothelial cell survival by interfering with apoptosis. In vitro and in vivo studies have established that regulation of apoptotic pathways may impact stroke outcome.21Mason R Leeds P Jacob R Hough C Zhang K Mason P Chuang D Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells.J Neurochem. 1999; 72: 1448-1456Crossref PubMed Scopus (93) Google Scholar In models of cerebral ischemia, inhibition or gene-inactivation of caspase-3 or caspase-1 results in mice that are partially resistant to ischemia,22Hara H Fink K Endres M Friedlander R Gagliardini V Yuan J Moskowitz M Attenuation of transient focal cerebral ischemic injury in transgenic mice expressing a mutant ICE inhibitory protein.J Cereb Blood Flow Metab. 1997; 17: 370-375Crossref PubMed Scopus (208) Google Scholar, 23Endres M Namura S Shimizu-Sasamata M Waeber C Zhang L Gomez-Isla T Hyman B Moskowitz M Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family.J Cerebral Blood Flow Metab. 1998; 18: 238-247Crossref PubMed Scopus (509) Google Scholar while gene transfer of inhibitor of apoptosis proteins (IAPs) in normal rats also attenuates ischemic damage.24Xu D Bureau Y McIntyre D Nicholson D Liston P Zhu Y Fong W Crocker S Korneluk R Robertson G Attenuation of ischemia-induced cellular and behavioral deficits by X chromosome-linked inhibitor of apoptosis protein overexpression in the rat hippocampus.J Neuroscience. 1999; 19: 5026-5033PubMed Google Scholar Survivin is a member of the IAP family, containing a single baculovirus IAP repeat (BIR) that facilitates interactions with caspase-3, caspase-7, and caspase-9.25Tamm I Wang Y Sausville E Scudiero DA Vigna N Oltersdorf T Reed JC IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs.Cancer Res. 1998; 58: 5315-5320PubMed Google Scholar Survivin has additional unique cell-survival properties, in that it is crucial for mitosis and cell-cycle progression.26Ambrosini G Adida C Altieri D A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3000) Google Scholar, 27Li F Ambrosini G Chu EY Plescia J Tognin S Marchisio PC Altieri DC Control of apoptosis and mitotic spindle checkpoint by survivin.Nature. 1998; 396: 580-584Crossref PubMed Scopus (1723) Google Scholar, 28Skoufias DA Mollinari C Lacroix FB Margolis RL Human survivin is a kinetochore-associated passenger protein.J Cell Biol. 2000; 151: 1575-1582Crossref PubMed Scopus (235) Google Scholar, 29Li F Ackermann EJ Bennett CF Rothermel AL Plescia J Tognin S Villa A Marchisio PC Altieri DC Pleiotropic cell-division defects and apoptosis induced by interference with survivin function.Nat Cell Biol. 1999; 1: 461-466Crossref PubMed Scopus (553) Google Scholar, 30Uren AG Wong L Pakusch M Fowler KJ Burrows FJ Vaux DL Choo KH Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype.Curr Biol. 2000; 10: 1319-1328Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar Notably, survivin is up-regulated in cultured vascular endothelial cells in response to angiogenic growth factors, including VEGF, angiopoietin-1, bFGF, and Plgf.6Tran J Rak J Sheehan C Saibil SD LaCasse E Korneluk RG Kerbel RS Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells.Biochem Biophys Res Commun. 1999; 264: 781-788Crossref PubMed Scopus (308) Google Scholar, 7O'Connor DS Schechner JS Adida C Mesri M Rothermel AL Li F Nath AK Pober JS Altieri DC Control of apoptosis during angiogenesis by survivin expression in endothelial cells.Am J Pathol. 2000; 156: 393-398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 31Harfouche R Hassessian HM Guo Y Faivre V Srikant CB Yancopoulos GD Hussain SN Mechanisms which mediate the antiapoptotic effects of angiopoietin-1 on endothelial cells.Microvasc Res. 2002; 64: 135-147Crossref PubMed Scopus (132) Google Scholar, 32Mesri M Morales-Ruiz M Ackermann EJ Bennett CF Pober JS Sessa WC Altieri DC Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting.Am J Pathol. 2001; 158: 1757-1765Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 33Adini A Kornaga T Firoozbakht F Benjamin LE Placental growth factor is a survival factor for tumor endothelial cells and macrophages.Cancer Res. 2002; 62: 2749-2752PubMed Google Scholar Furthermore, while survivin expression is detected in several organs during development, it is prominent in the fetal brain,34Adida C Crotty P McGrath J Berrebi D Diebold J Altieri D Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation.Am J Pathol. 1998; 152: 43-49PubMed Google Scholar maintaining expression into adulthood, where it is present in the choroid plexus, ependymal cells, neurons, astrocytes, and oligodendrocytes.35Sasaki T Lopes MB Hankins GR Helm GA Expression of survivin, an inhibitor of apoptosis protein, in tumors of the nervous system.Acta Neuropathol (Berl). 2002; 104: 105-109Crossref PubMed Scopus (88) Google Scholar In view of the unique cell survival properties of survivin, its expression in ischemia-sensitive regions of the brain, and most notably its differential regulation in endothelial cells in response to VEGF and other angiogenic factors, we used a mouse model of stroke to test the hypothesis that survivin plays a crucial role in neovascularization and repair after cerebrovascular occlusion. Generation of survivin+/− mice by homologous recombination in embryonic stem (ES) cells has been previously reported.36Conway EM Pollefeyt S Steiner-Mosonyi M Luo W DeVriese A Lupu F Bono F Leducq N Dol F Schaeffer P Collen D Herbert J-M Deficiency of survivin in transgenic mice exacerbates Fas-induced apoptosis via mitochondrial pathways.Gastroenterology. 2002; 123: 619-631Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar For the current studies, transgenic mice were bred onto a Swiss:129s (∼94:6) background. These animals express ∼50% levels of survivin mRNA, and respond with increased sensitivity to FasL-induced hepatic apoptosis. No other phenotypic abnormalities have been identified. Survivin+/+ littermates were used as controls for experiments on survivin+/− mice. In other experiments to evaluate temporal changes in response to stroke, BALB/c mice were used. Focal cerebral ischemia was achieved by permanent occlusion of the MCA according to established methods.37Nagai N De Mol M Lijnen H Carmeliet P Collen D Role of plasminogen system components in focal cerebral ischemic infarction.Circulation. 1999; 99: 2440-2444Crossref PubMed Scopus (212) Google Scholar Briefly, 10- to 12-week-old mice weighing 20 to 30 g were anesthetized by intraperitoneal injection of ketamine and xylazine. Atropine (1 mg/kg) was administered intramuscularly, and body temperature was maintained at 37°C. A U-shaped incision was made between the left ear and left eye. The cranial and dorsal segments of the temporalis muscle were transected and retracted, thereby exposing the skull. A 2-mm-diameter opening was made in the region over the MCA with a handheld drill, with saline superfusion to prevent heat injury. The meninges were removed with a forceps, and the MCA was occluded with three adjacent ligations with 10−0 nylon thread (Ethicon, Johnson and Johnson, Dilbeek, Belgium). Finally, the artery was transected distal to the ligation. The temporalis muscle and skin were reconstructed, and the mice were allowed to recover. Control (sham) mice were treated identically, excluding ligation of the MCA. Mortality during the procedure did not exceed 10%. At different times (6 hours to 7 days) after MCAO, the mice were re-anesthetized and the brains were quickly removed and either fixed in 4% paraformaldehyde for 3 hours at 4°C and embedded in paraffin for subsequent histological analyses, or the intact brain was cut into slices and immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) in saline for quantitation of infarct volume (see below). In each case, a minimum of 4 to 7 mice was used at each interval. For histological analyses, 7-μm brain sections were cut. Sections were dewaxed, rehydrated, antigen retrieval was performed, and they were incubated with proteinase K (20 mg/ml Tris-HCl, pH7.5) for 30 minutes at 37°C. Immunoperoxidase staining was performed according to standard techniques using the following antibodies: Specific rabbit anti-murine survivin antibodies were generated as described.38Conway EM Pollefeyt S Cornelissen J DeBaere I Steiner-Mosonyi M Ong K Baens M Collen D Schuh AC Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions.Blood. 2000; 95: 1435-1442Crossref PubMed Google Scholar Rabbit anti-rat thrombomodulin (anti-TM) antibodies were a gift from Dr. R.W. Jackman (Harvard University, Boston, MA). All other antibodies were obtained from SanverTECH (Boechout, Belgium). Vessel density was quantified by visually counting TM-staining vessels in 5 non-adjacent (>100 μm distant) cross-sections per mouse (n = 4) in at least 4 high-power fields per section,13Beck H Acker T Wiessner C Allegrini PR Plate KH Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat.Am J Pathol. 2000; 157: 1473-1483Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar and dividing by the measured areas. Results are expressed as number of vessels/mm2. The selected areas encompassed the entire penumbra and central infarct region. The leptomeninges, which is usually a site of increased vascularity postischemia, was excluded because the entire leptomeninges was not uniformly intact in all specimens after processing for immunohistologic analyses. All assessments were performed by one blinded microscopist. The mean value from each mouse was used to calculate the overall mean ± SEM. Paraffin-embedded brain sections were processed as above, and co-incubated with primary endothelial cell-specific goat anti-Glut139Golden PL Pardridge WM Brain microvascular P-glycoprotein and a revised model of multidrug resistance in brain.Cell Mol Neurobiol. 2000; 20: 165-181Crossref PubMed Scopus (70) Google Scholar antibodies (Santa Cruz Biotechnology, SanverTECH) and biotinylated rabbit anti-survivin antibodies, or combinations of the corresponding pre-immune antibodies. After washes, sections were further incubated with rhodamine-conjugated anti-goat antibodies and FITC-conjugated streptavidin. Analyses were performed using a confocal laser-scanning microscope (Leica, Wetzlar, Germany). Settings to exclude non-specific background were determined by using the corresponding primary pre-immune antibodies. After surgical removal of the brain and immersion of 1-mm slices in TTC, the stained sections were photographed, and the well-defined necrotic and apparently viable areas were quantified by planimetry, after which the infarct volume was calculated.40Lin T He Y Wu G Khan M Hsu C Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats.Stroke. 1993; 24: 117-121Crossref PubMed Scopus (629) Google Scholar The intact contralateral hemispheric volume was also determined, and there was ∼2.5% intermouse variability within the same strain of mice. Thus, absolute infarct volumes were used for the purposes of comparison. Total RNA was isolated from homogenized tissue in TRIzol reagent (Life Technologies, Merelbeke, Belgium) according to the manufacturer's instructions. Standard curves for quantitative real-time polymerase chain reaction (PCR) were generated from plasmids containing cDNAs encoding murine survivin140, VEGF, Plgf, HIF1α, HIF2α, and p53. Concurrent PCR to detect expression of the reference gene HPRT, using primers HPRTF (sense 5′ ttatcagactgaagagctactgtaatgatc) and HPRTR (antisense 5′ ttaccagtgtcaattatatcttcaacaatc) and probe 5′-(JOE)-tgagagatcatctccaccaataacttttatgtccc-(TAMRA)−3′) allowed for relative quantitation of expression of gene transcripts as a function of HPRT copy number. Real-time PCR measurements were done in triplicate, with duplicate or triplicate samples under the same condition. Entire experiments were performed a minimum of two times. Cells were washed and lysed on ice in a solution containing 0.5% Triton X-100, 50 mmol/L NaCl, 5 mmol/L ethylenediaminetetraacetate, 10 mmol/L Tris-HCl pH7.5 in the presence of protease inhibitors. 100 μg were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions and transferred to a nylon filter which was blocked with 3% bovine serum albumin in TBS with 0.01% Tween and incubated for 2 to 18 hours with the primary antibody. After washing and incubation of the filter with the appropriate secondary antibody conjugated to horseradish peroxidase, detection was accomplished using the enhanced chemiluminescence method (Amersham-Pharmacia, Freiburg, Denmark). Equivalent loading was confirmed by re-blotting the filters for detection of actin. A regulated hypoxia chamber, maintained at room temperature, was used to expose mice to an ambient oxygen tension of 5.5% overnight. Mice were immediately sacrificed for analyses subsequent to hypoxia. Oxygen tensions were continuously monitored during experiments, and there was less than 1% variation from the target in the recorded oxygen tension during any experiment. ES cells lacking both alleles for the genes encoding VEGF and HIF1α, with corresponding wild-type control cells, were generated at the Center for Transgene Technology and Gene Therapy by high G418 selection of ES cells heterozygous for each gene. ES cells were routinely cultured on fibroblast feeders and in the presence of leukemia inhibitory factor to prevent differentiation. Human umbilical vein endothelial cells (HUVECs) were cultured as previously described.41Conway E Nowakowski B Steiner-Mosonyi M Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation.Blood. 1992; 80: 1254-1263Crossref PubMed Google Scholar Before hypoxia exposure, cells were split onto gelatin-coated plates, refed with complete media, and 24 hours later, incubated in a humidified hypoxia chamber for 20 hours before processing for RNA or Western blot analyses. An oxygen tension of 2% (5% CO2, balance N2) was selected due to reported effects on transcription of angiogenesis-related genes in endothelial cells under similar conditions.42Jung F Haendeler J Hoffmann J Reissner A Dernbach E Zeiher AM Dimmeler S Hypoxic induction of the hypoxia-inducible factor is mediated via the adaptor protein Shc in endothelial cells.Circ Res. 2002; 91: 38-45Crossref PubMed Scopus (48) Google Scholar, 43Christensen RA Fujikawa K Madore R Oettgen P Varticovski L NERF2, a member of the Ets family of transcription factors, is increased in response to hypoxia and angiopoietin-1: a potential mechanism for Tie2 regulation during hypoxia.J Cell Biochem. 2002; 85: 505-515Crossref PubMed Scopus (33) Google Scholar Studies were performed at least twice, with different ES cell clones, and real-time PCR was done in triplicate. Animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Leuven. Statistical analyses of data were conducted with the StatView computer program (Abacus Concepts Inc., Berkeley, CA) or with InStat 3 (MacKiev Company, Cupertino, CA). Student's t-test was used to compare groups in which the standard deviations were not significantly different. The data are represented as the mean ± SE. Using specific anti-survivin antibodies for immunoperoxidase staining of sections of fixed brain tissues, we first examined the expression pattern of survivin in the brains of normal adult BALB/c mice. Low levels of survivin were detected in neurons within the CA-1, CA-2, and CA-3 regions of the hippocampus, the dentate gyrus, and the pyramidal cells of the cortex (Figure 1, A to C), as similarly reported.35Sasaki T Lopes MB Hankins GR Helm GA Expression of survivin, an inhibitor of apoptosis protein, in tumors of the nervous system.Acta Neuropathol (Berl). 2002; 104: 105-109Crossref PubMed Scopus (88) Google Scholar Survivin was not otherwise detectable in any other sites. Specifically, there was no expression in or around the vasculature. In adjacent sections, no signal was detectable when pre-immune Ig was used (Figure 1D), or when the primary antibody was excluded (not shown). Survivin expression in response to stroke was evaluated by using a murine model of permanent focal cerebral ischemia in which the MCA was ligated. Adult BALB/c mice were used because in pilot studies cerebral infarct volumes were consistently larger and there was less mouse-to-mouse variability than in other strains (eg, Swiss, 129s, C57Bl6). Demarcation of infarcts was readily identified by hematoxylin and eosin (H&E) staining (Figure 1E) as nuclei of cells within the infarct were condensed, and the cells retracted. From 6 hours to 7 days after MCAO, the expression of survivin remained detectable in both cerebral hemispheres in the neurons of the hippocampus, dentate gyrus, and cortex. The pattern and intensity of staining was similar to tha
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