Bivalirudin versus unfractionated heparin during percutaneous coronary intervention

BACKGROUND: Whether bivalirudin is superior to unfractionated heparin in patients with stable or unstable angina who undergo percutaneous coronary intervention (PCI) after pretreatment with clopidogrel is unknown. METHODS: We enrolled 4570 patients with stable or unstable angina (with normal levels of troponin T and creatine kinase MB) who were undergoing PCI after pretreatment with a 600-mg dose of clopidogrel at least 2 hours before the procedure; 2289 patients were randomly assigned in a double-blind manner to receive bivalirudin, and 2281 to receive unfractionated heparin. The primary end point was the composite of death, myocardial infarction, urgent target-vessel revascularization due to myocardial ischemia within 30 days after randomization, or major bleeding during the index hospitalization (with a net clinical benefit defined as a reduction in the incidence of the end point). The secondary end point was the composite of death, myocardial infarction, or urgent target-vessel revascularization. RESULTS: The incidence of the primary end point was 8.3% (190 patients) in the bivalirudin group as compared with 8.7% (199 patients) in the unfractionated-heparin group (relative risk, 0.94; 95% confidence interval [CI], 0.77 to 1.15; P=0.57). The secondary end point occurred in 134 patients (5.9%) in the bivalirudin group and 115 patients (5.0%) in the unfractionated-heparin group (relative risk, 1.16; 95% CI, 0.91 to 1.49; P=0.23). The incidence of major bleeding was 3.1% (70 patients) in the bivalirudin group and 4.6% (104 patients) in the unfractionated-heparin group (relative risk, 0.66; 95% CI, 0.49 to 0.90; P=0.008). CONCLUSIONS: In patients with stable and unstable angina who underwent PCI after pretreatment with clopidogrel, bivalirudin did not provide a net clinical benefit (i.e., it did not reduce the incidence of the composite end point of death, myocardial infarction, urgent target-vessel revascularization, or major bleeding) as compared with unfractionated heparin, but it did significantly reduce the incidence of major bleeding. (ClinicalTrials.gov number, NCT00262054.)

Sphingosine kinase 1 and 2 regulate the capacity of mesangial cells to resist apoptotic stimuli in an opposing manner

Abstract Sphingosine kinases (SKs) are key enzymes regulating the production of sphingosine-1-phosphate (S1P), which determines important cell responses including cell growth and death. Here we show that renal mesangial cells isolated from wild-type, SK-1(-/-), and SK-2(-/-) mice show a differential response to apoptotic stimuli. Wild-type mesangial cells responded to staurosporine with increased DNA fragmentation and caspase-3 processing, which was enhanced in SK-1(-/-) cells. In contrast, SK-2(-/-) cells were highly resistant to staurosporine-induced apoptosis. Furthermore, the basal phosphorylation and activity of the anti-apoptotic protein kinase B (PKB) and of its substrate Bad were decreased in SK-1(-/-) but not in SK-2(-/-) cells. Upon staurosporine treatment, phosphorylation of PKB and Bad decreased in wild-type and SK-1(-/-) cells, but remained high in SK-2(-/-) cells. In addition, the anti-apoptotic Bcl-X(L) was significantly upregulated in SK-2(-/-) cells, which may further contribute to the protective state of these cells. In summary, our data show that SK-1 and SK-2 have opposite effects on the capacity of mesangial cells to resist apoptotic stimuli. This is due to differential modulation of the PKB/Bad pathway and of Bcl-X(L) expression. Thus, subtype-selective targeting of SKs will be critical when considering these enzymes as therapeutic targets for the treatment of inflammation or cancer.

New players on the center stage: sphingosine 1-phosphate and its receptors as drug targets

The recent identification of a cellular balance between ceramide and sphingosine 1-phosphate (S1P) as a critical regulator of cell growth and death has stimulated increasing research effort to clarify the role of ceramide and S1P in various diseases associated with dysregulated cell proliferation and apoptosis. S1P acts mainly, but not exclusively, by binding to and activating specific cell surface receptors, the so-called S1P receptors. These receptors belong to the class of G protein-coupled receptors that constitute five subtypes, denoted as S1P(1)-S1P(5), and represent attractive pharmacological targets to interfere with S1P action. Whereas classical receptor antagonists will directly block S1P action, S1P receptor agonists have also proven useful, as recently shown for the sphingolipid-like immunomodulatory substance FTY720. When phosphorylated by sphingosine kinase to yield FTY720 phosphate, it acutely acts as an agonist at S1P receptors, but upon prolonged presence, it displays antagonistic activity by specifically desensitizing the S1P(1) receptor subtype. This commentary will cover the most recent developments in the field of S1P receptor pharmacology and highlights the potential therapeutic benefit that can be expected from these novel drug targets in the future.

Sphingosine kinase-1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells

Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1alpha and HIF-2alpha, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1alpha or HIF-2alpha by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression.

Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

International Common Otology Database: taste disturbance after stapes surgery

OBJECTIVE: To use a study on dysgeusia to assess the usefulness of an otology database. STUDY DESIGN: Data were extracted from the international Common Otology Database. INTERVENTION: Primary stapes operations. MAIN OUTCOME MEASURE AND RESULTS: From a cohort of 14 otologists, only 8 (57%) were able to satisfy external validation and maintain data input for a period of at least 6 months. The rates of dysgeusia varied from 0 to 39% at 3 months and 0 to 27% at 6 months. The percentages of patients with taste disturbance at 6 months in the "nerve-cut" and "nerve-preserved" groups were 22.7 and 10.9%, respectively, although this was not statistically significant (chi2; p = 0.325). CONCLUSION: Many surgeons found it difficult to maintain a prospective otology database. The rates of certain subjective symptoms such as dysgeusia are influenced by how vigorously the reviewers prompt the response from the patients. Dysgeusia after stapes surgery is common even if the chorda tympani nerve is preserved. Many patients whose chorda tympani nerve is divided may not complain of dysgeusia.

IL-6 transsignalling modulates the early effector phase of EAE and targets the blood-brain barrier

Interleukin-6 (IL-6) plays a crucial role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). It exerts its cellular effects by a membrane-bound IL-6 receptor (IL-6R), or, alternatively, by forming a complex with the soluble IL-6R (sIL-6R), a process named IL-6 transsignalling. Here we investigate the role of IL-6 transsignalling in myelin basic protein (MBP)-induced EAE in the Lewis rat. In vivo blockade of IL-6 transsignalling by the injection of a specifically designed gp130-Fc fusion protein significantly delayed the onset of adoptively transferred EAE in comparison to control rats injected with PBS or isotype IgG. Histological evaluation on day 3 after immunization revealed reduced numbers of T cells and macrophages in the lumbar spinal cord of gp130-Fc treated rats. At the same time, blockade of IL-6 transsignalling resulted in a reduced expression of vascular cell adhesion molecule-1 on spinal cord microvessels while experiments in cell culture failed to show a direct effect on the regulation of endothelial adhesion molecules. In experiments including active EAE and T cell culture, inhibition of IL-6 transsignalling mildly increased T cell proliferation, but did not change severity of active MBP-EAE or regulate Th1/Th17 responses. We conclude that IL-6 transsignalling may play a role in autoimmune inflammation of the CNS mainly by regulating early expression of adhesion molecules, possibly via cellular networks at the blood-brain barrier.

Radiation metabolomics. 1. Identification of minimally invasive urine biomarkers for gamma-radiation exposure in mice

Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for gamma-radiation biodosimetry in a mouse model. Mice were gamma-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and beta-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose-response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to gamma radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.

Overexpression of lymphotoxin in T cells induces fulminant thymic involution

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

Radiation metabolomics. 2. Dose- and time-dependent urinary excretion of deaminated purines and pyrimidines after sublethal gamma-radiation exposure in mice

Gamma-radiation exposure of humans is a major public health concern as the threat of terrorism and potential hostile use of radiological devices increases worldwide. We report here the effects of sublethal gamma-radiation exposure on the mouse urinary metabolome determined using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry-based metabolomics. Five urinary biomarkers of sublethal radiation exposure that were statistically significantly elevated during the first 24 h after exposure to doses ranging from 1 to 3 Gy were unequivocally identified by tandem mass spectrometry. These are deaminated purine and pyrimidine derivatives, namely, thymidine, 2'-deoxyuridine, 2'-deoxyxanthosine, xanthine and xanthosine. Furthermore, the aminopyrimidine 2'-deoxycytidine appeared to display reduced urinary excretion at 2 and 3 Gy. The elevated biomarkers displayed a time-dependent excretion, peaking in urine at 8-12 h but returning to baseline by 36 h after exposure. It is proposed that 2'-deoxyuridine and 2'-deoxyxanthosine arise as a result of gamma irradiation by nitrosative deamination of 2'-deoxycytidine and 2'-deoxyguanosine, respectively, and that this further leads to increased synthesis of thymidine, xanthine and xanthosine. The urinary excretion of deaminated purines and pyrimidines, at the expense of aminopurines and aminopyrimidines, appears to form the core of the urinary radiation metabolomic signature of mice exposed to sublethal doses of ionizing radiation.

Sirolimus-eluting stent treatment at high-volume centers confers lower mortality at 6-month follow-up: results from the prospective multicenter German Cypher Registry

BACKGROUND: Studies continue to identify percutaneous coronary intervention procedural volume both at the institutional level and at the operator level as being strongly correlated with outcome. High-volume centers have been defined as those that perform >400 percutaneous coronary intervention procedures per year. The relationship between drug-eluting stent procedural volume and outcome is unknown. We investigated this relationship in the German Cypher Registry. METHODS AND RESULTS: The present analysis included 8201 patients treated with sirolimus-eluting stents between April 2002 and September 2005 in 51 centers. Centers that recruited >400 sirolimus-eluting stent patients in this time period were considered high-volume centers; those with 150 to 400 patients were considered intermediate-volume centers; and those with <150 patients were designated as low-volume centers. The primary end point was all death, myocardial infarction, and target-vessel revascularization at 6 months. This end point occurred in 11.3%, 12.1%, and 9.0% of patients in the low-, intermediate-, and high-volume center groups, respectively (P=0.0001). There was no difference between groups in the rate of target-vessel revascularization (P=0.2) or cerebrovascular accidents (P=0.5). The difference in death/myocardial infarction remained significant after adjustment for baseline factors (odds ratio 1.85, 95% confidence interval 1.31 to 2.59, P<0.001 for low-volume centers; odds ratio 1.69, 95% confidence interval 1.29 to 2.21, P<0.001 for intermediate-volume centers). Patient and lesion selection, procedural features, and postprocedural medications differed significantly between groups. CONCLUSIONS: The volume of sirolimus-eluting stent procedures performed on an institutional level was inversely related to death and myocardial infarction but not to target-vessel revascularization at 6-month follow-up. Safety issues are better considered in high-volume centers. These findings have important public health policy implications.