Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0022116 (ischemia)
91,303 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Catheter-based percutaneous transluminal gene delivery (PTGD) into the coronary artery still falls behind the expectations of an efficient myocardial gene delivery system. In this study gene delivery was applied by selective pressure-regulated retroinfusion through the coronary veins to prolong adhesion of replication defective adenovirus within the targeted myocardium. Adenoviral vectors consisted either of luciferase (Ad.rsv-Luc) or beta-galactosidase (Ad.rsv-betaGal) reporter gene under control of an unspecific promotor derived from the Rous sarcoma virus (RSV). In this pig model, selective retrograde gene delivery into the anterior cardiac vein during a brief period of ischemia substantially increased reporter gene expression in the targeted myocardium (LAD region) compared with antegrade delivery as a control. Repeated retrograde delivery during two periods of brief ischemia resulted in a more homogeneous transmural expression predominantly observed in cardiomyocytes (X-gal-staining). In the nontargeted myocardium (CX region) there was no evidence for adenoviral transfection. From our data we infer that selective pressure-regulated retroinfusion is a promising approach for efficient percutaneous transluminal gene delivery to the myocardium. Gene Therapy (2000) 7, 232-240.
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PMID:Myocardial gene transfer by selective pressure-regulated retroinfusion of coronary veins. 1069

Previous studies have indicated that advanced age is associated with impaired angiogenesis in part because of reduced levels of vascular endothelial growth factor (VEGF) expression. To investigate potential mechanisms responsible for this age-dependent defect in VEGF expression, aortic smooth muscle cells isolated from young rabbits (ages 6-8 months) or old rabbits (ages 4-5 years) were exposed to normoxic (21% oxygen) or hypoxic (0.1% oxygen) conditions. Hypoxia-induced VEGF expression was significantly lower in old versus young cells. VEGF mRNA stability in hypoxic conditions was similar in both young and old cells. However, transient transfection with a luciferase reporter gene that was transcriptionally regulated by the VEGF promoter revealed a significant defect in VEGF up-regulation following hypoxia in old versus young cells (a 43 versus 117% increase in luciferase activity, p < 0.05); this difference was not seen when a deletion construct lacking the hypoxia-inducible 1 (HIF-1) binding site was used. Moreover, although HIF-1 alpha-mRNA expression was shown to be similar in young and old smooth muscle cells, HIF-1 alpha protein and DNA binding activity were significantly reduced in old versus young smooth muscle cells that were exposed to hypoxia. We propose that age-dependent reduction in hypoxia-induced VEGF expression results from reduced HIF-1 activity and may explain the previously described age-dependent impairment of angiogenesis in response to ischemia.
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PMID:Age-dependent defect in vascular endothelial growth factor expression is associated with reduced hypoxia-inducible factor 1 activity. 1088 14

Vascular endothelin-1 (ET-1) levels are elevated in patients with renal allograft rejection, and the mitogenic and pressor actions of ET-1 might contribute to transplant vasculopathy, posttransplantation hypertension, and ischemia-reperfusion injury. In contrast, relatively little is known about tubular expression of ET-1 in acute or chronic rejection of renal allografts. We sought to determine whether tubular ET-1 levels were altered in patients with acute or chronic renal allograft rejection. Immunohistochemical analysis of tubular ET-1 was performed in renal biopsy specimens from 18 patients with acute rejection, 7 patients with chronic rejection, and 5 normal kidneys excised for localized neoplasm. The diagnosis of acute or chronic rejection in each patient was verified and graded using the Banff schema. Renal tubular epithelium from patients with allograft rejection had markedly elevated staining for ET-1 compared with normal kidneys. Tubular ET-1 levels were elevated in 18 of 18 patients with acute rejection and 5 of 7 patients with chronic rejection. Tubular ET-1 staining was graded from 0 to +3 as follows: normal kidneys, 1.2 +/- 0.2; acute rejection, 2.3 +/- 0.4 (P < 0.01); and chronic rejection, 2.2 +/- 0.5 (P < 0.01). ET-1 staining was prominent in both proximal and distal tubules, and we observed abundant ET-1 secretion from proximal tubular epithelium in culture. Moreover, ET-1 activated the c-fos immediate early gene promoter in proximal tubular cells transfected with a c-fos luciferase reporter. We conclude that elevated tubular ET-1 levels are associated with acute and chronic rejection of renal allografts. Our results also suggest distinct pathophysiological roles for the tubular and vascular ET-1 systems in renal allograft rejection.
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PMID:Elevated endothelin-1 in tubular epithelium is associated with renal allograft rejection. 1097 86

Conventional large (500-800 nm) multilamellar liposomes encapsulating DNA have been used in vivo as gene vectors into rat and pig liver. By using the intraportal vein route, high dose DNA (10 mg/kg) provided low efficiency and transient luciferase gene expression in the liver. This gene expression was, however, increased by liver resection (> 50%), ischemia (20 min) or orthotopic transplantation. As evidenced by histochemical analysis of beta-galactosidase expression, the gene transfection mainly ensued in Kupffer cells, but spleen and lung were contaminated. In comparison, injection into the bile duct of even 25-fold lower dose of liposome-encapsulated DNA (0.4 mg/kg) produced higher (100-fold) and long-lasting (during 6 days, at least) luciferase expression in rat liver. The gene expression was restricted to the liver and enhanced by liver resection. By this route, transgene-expressing cells were mainly hepatocytes. A treatment with colchicine prior to the administration of the vector allowed the persistence of relative high gene expression for at least 7 days. In pigs, qualitatively similar, but quantitatively less efficient gene expression was obtained by either the portal vein or the bile duct route. These results indicate that intrabile duct route might render large non-viral vectors applicable to gene transfer into the hepatocytes. The efficiency of liposome-mediated gene transfer into the liver can be increased by liver resection, ischemia or transplantation performed before DNA injection.
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PMID:In vivo liver-directed gene transfer in rats and pigs with large anionic multilamellar liposomes: routes of administration and effects of surgical manipulations on transfection efficiency. 1114 37

Pharmacological uncoupling of mitochondrial oxidation from phosphorylation promotes preconditioning-like cardioprotection in the isolated rat heart. We hypothesized that modest mitochondrial uncoupling may be a critical cellular event in orchestrating preconditioning. Human-derived Girardi cells and murine C2C12 skeletal myotubes were preconditioned using simulated ischemia, adenosine, and diazoxide. Cell viability after 6 hours of simulated ischemia was measured using lactate dehydrogenase release and propidium iodide uptake. Mitochondrial inner membrane potential (DeltaPsim) was investigated by flow cytometry, cellular ATP by recombinant firefly-luciferase bioluminescence, and cellular oxygen consumption using oximetry. Preconditioning enhanced cell viability with attenuation of lactate dehydrogenase release (>/=30%, P<0.05 versus ischemic controls) and a reduction in propidium iodide uptake by >/=26% versus ischemic controls after simulated ischemia in both cell lines. In Girardi cells, preconditioning induced the following phenotype immediately before index ischemia: (1) decreased DeltaPsim (JC-1: simulated ischemia 90+/-3%, adenosine 82+/-7%, diazoxide 87+/-4%, versus control 100%, P<0.05); (2) attenuation in cellular ATP levels (CTL 0.21+/-0.03 nmol/L ATP/microg protein, simulated ischemia 0.12+/-0.02, adenosine 0.15+/-0.02, diazoxide 0.11+/-0.02, P<0.05); and (3) enhanced cellular oxygen consumption (control 2.3+/-0.1 nmol/L oxygen/min/1x10(6) cells, simulated ischemia 3.1+/-0.1, adenosine 3.1+/-0.3, diazoxide 2.6+/-0.2, P<0.05). Cytoprotection, mitochondrial depolarization, and enhanced oxygen consumption were attenuated by the putative mitochondrial K(ATP)-channel antagonist 5-hydroxydecanoate. The uncoupled phenotype in response to preconditioning was similarly observed in C2C12 myotubes. The present study suggests that modest mitochondrial uncoupling represents a unifying cellular response which may be important in directing preconditioning-mediated cytoprotection.
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PMID:Ischemic and pharmacological preconditioning in Girardi cells and C2C12 myotubes induce mitochondrial uncoupling. 1167 1

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.
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PMID:Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. 1196 Mar 13

We have previously demonstrated that cardioprotection induced by the infusion of a selective delta1-opioid agonist is mediated by the specific translocation of PKC-delta to the mitochondria in in vivo rat hearts and via opening of the mitochondrial KATP channel. Ischemic preconditioning (IPC) is also thought to involve the translocation of specific isoforms of PKC and KATP channel activation. Therefore, we utilized the PKC-delta selective antagonist, rottlerin, to assess the effect of inhibition of this isozyme on cardioprotection induced by one-cycle of IPC prior to 30 minutes of ischemia and 2 hours of reperfusion. Infarct size (IS) was determined by tetrazolium chloride staining and expressed as a percent of the area at risk (AAR). Non-preconditioned control animals had an IS/AAR of 59.7 +/- 1.6. IPC significantly reduced the extent of myocardial infarction (6.3 +/- 1.4). Rottlerin, 0.3 mg/kg, did not alter IS/AAR in control animals (55.0 +/- 5.6), and had no significant effect on IS/AAR in preconditioned animals (14.4 +/- 3.8). Additionally, we demonstrated, using a luciferase-based assay to determine the rate of ATP synthesis and state of mitochondrial bioenergetics, that IPC preserves ATP synthesis in the ischemic myocardium and that this preservation is attenuated by the isoform non-selective PKC inhibitor, chelerythrine, but not by the delta-selective antagonist, rottlerin. These data suggest that PKC-delta does not play an important role in IPC and that differences in isoform importance are evident during pharmacological versus ischemia-induced preconditioning.
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PMID:PKC-delta inhibition does not block preconditioning-induced preservation in mitochondrial ATP synthesis and infarct size reduction in rats. 1199 77

Geldanamycin (GA), a benzoquinone ansamycin, binds Hsp90 in vitro, releases heat shock factor (HSF1) and induces heat shock proteins (Hsps). Because viral and transgenic overexpression of Hsps protects cells against ischemia in vitro, we hypothesized that GA would protect brain from focal ischemia by inducing Hsps in vivo. Adult male Sprague-Dawley rats were subjected to 2-hour middle cerebral artery occlusions (MCAO) using the suture technique followed by 22-h reperfusions. GA or vehicle was injected into the lateral cerebral ventricles (i.c.v) 24 h before ischemia. Geldanamycin at 1 microg/kg decreased infarct volumes by 55.7% (p < 0.01) and TUNEL-positive cells by 30% in cerebral cortex. GA also improved behavioral outcomes (p < 0.01) and reduced brain edema (p < 0.05). Western blots showed that the 1 microg/kg GA dose induced Hsp70 and Hsp25 protein 8.2-fold and 2.7-fold, respectively, by 48 h following administration. Immunocytochemistry showed that GA induced Hsp70 in neurons and Hsp25 in glia and arteries in cortex, hippocampus, hypothalamus, and other brain regions. GA reduced co-immunoprecipitation of HSF1 with Hsp90 in brain tissue homogenates, promoted HSE-binding of HSF in brain nuclear extracts using gel shift assays, and increased luciferase reporter gene transcription for the Hsp70 promoter in PC12 cells. The data show that geldanamycin protects brain from focal ischemia and that this may be due, at least in part, to geldanamycin stimulation of heat shock gene transcription.
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PMID:Geldanamycin induces heat shock proteins in brain and protects against focal cerebral ischemia. 1206 83

Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.
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PMID:Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion. 1210 62

Renal ischemia is the result of a complex series of events, including decreases in oxygen supply (hypoxia) and the availability of cellular energy (ATP depletion). In this study, the functional activation of two stress-responsive transcription factors, i.e., heat shock factor-1 (HSF-1) and hypoxia-inducible factor-1 (HIF-1), in the kidney was assessed. When rats were subjected to 45 min of renal ischemia, electrophoretic mobility shift assays of kidney nuclear extracts revealed rapid activation of both HIF-1 and HSF. Western blot analyses further demonstrated that this activation resulted in increased expression of the HSF and HIF-1 target genes heat shock protein-72 and heme oxygenase-1, respectively. Whether hypoxia or ATP depletion alone could produce similar activation patterns in vitro was then investigated. Renal epithelial LLC-PK(1) cells were subjected to either ATP depletion (0.1 microM antimycin A and glucose deprivation) or hypoxia (1% O(2)). After ATP depletion, HSF was rapidly activated (within 30 min), whereas HIF-1 was unaffected. In contrast, hypoxia led to the activation of HIF-1 but not HSF. Hypoxic activation of HIF-1 was observed within 30 min and persisted for 4 h, whereas no HSF activation was detected even with prolonged periods of hypoxia. HIF-1 was transcriptionally active in LLC-PK(1) cells, as demonstrated by luciferase reporter gene assays using the vascular endothelial growth factor promoter or a synthetic promoter construct containing three hypoxia-inducible elements. Interestingly, intracellular ATP levels were not affected by hypoxia but were significantly reduced by ATP depletion. These findings suggest that HIF-1 is activated specifically by decreased O(2) concentrations and not by reduced ATP levels alone. In contrast, HSF is activated primarily by metabolic stresses associated with ATP depletion and not by isolated O(2) deprivation. In vivo, the two transcription factors are simultaneously activated during renal ischemia, which might account for observed differences between in vivo and in vitro epithelial cell injury and repair. Selective modulation of either pathway might therefore be of potential interest for modification of the response of the kidney to ischemia, as well as the processes involved in recovery from ischemia.
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PMID:Functional activation of heat shock factor and hypoxia-inducible factor in the kidney. 1213 41


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