Endothelial NOS (NOS3) impairs myocardial function in developing sepsis

Endothelial nitric oxide synthase (NOS)3-derived nitric oxide (NO) modulates inotropic response and diastolic interval for optimal cardiac performance under non-inflammatory conditions. In sepsis, excessive NO production plays a key role in severe hypotension and myocardial dysfunction. We aimed to determine the role of NOS3 on myocardial performance, NO production, and time course of sepsis development. NOS3(−/−) and C57BL/6 wildtype mice were rendered septic by cecum ligation and puncture (CLP). Cardiac function was analyzed by serial echocardiography, in vivo pressure and isolated heart measurements. Cardiac output (CO) increased to 160 % of baseline at 10 h after sepsis induction followed by a decline to 63 % of baseline after 18 h in wildtype mice. CO was unaltered in septic NOS3(−/−) mice. Despite the hyperdynamic state, cardiac function and mean arterial pressure were impaired in septic wildtype as early as 6 h post CLP. At 12 h, cardiac function in septic wildtype was refractory to catecholamines in vivo and respective isolated hearts showed impaired pressure development and limited coronary flow reserve. Hemodynamics remained stable in NOS3(−/−) mice leading to significant survival benefit. Unselective NOS inhibition in septic NOS3(−/−) mice diminished this survival benefit. Plasma NO(x)- and local myocardial NO(x)- and NO levels (via NO spin trapping) demonstrated enhanced NO(x)- and bioactive NO levels in septic wildtype as compared to NOS3(−/−) mice. Significant contribution by inducible NOS (NOS2) during this early phase of sepsis was excluded. Our data suggest that NOS3 relevantly contributes to bioactive NO pool in developing sepsis resulting in impaired cardiac contractility. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00395-013-0330-8) contains supplementary material, which is available to authorized users

Die Bedeutung der NO-Synthasen im murinen Modell der septischen Hämodynamik

Sepsis is one of the biggest challenges for modern medicine. The mortality rate is very high, the incidence rate is rising worldwide and the cost for the health sector is enormous. A crucial share in the pathophysiology must be attributed to cardiocirculatory failure triggered by a generalised vasodilatation as well as septic cardiomyopathy. An important mediator for these two phenomena is nitrogen monoxide. It was the object of the present dissertation to examine the nitrogen-oxide-mediated effects on septic haemodynamics in a murine CLP model and to clarify the significance attributable to the respective NO synthases in this connection. To this end, the following experiments were conducted in wild-type and eNOS-/- mice with and without a selective blockade of the inducible and neuronal NO synthase: In order to quantify the formation of nitrogen monoxide, the nitrite and nitrate levels were measured basally and after the induction of sepsis both in the plasma and in the cardiac tissue. The cardiovascular function was examined before and after the application of noradrenaline by means of invasive pressure/volume measurement via a miniature catheter. In order to measure the effect of nitrogen monoxide on the myocardial inflammatory reaction, a histological analysis of the infiltration of leucocytary cells into the cardiac muscle tissue was conducted. Likewise, the sepsis-modulated expression rate of the iNOS was measured by means of quantitative RT-PCR and in a separate population, the mean survival time of the various groups of laboratory animals was determined. Our results show that, in septic animals, there was a severe increase in the plasma NOx level as well as in the inflammatory reaction of the cardiac muscle. The consequence was a pronounced cardiovascular dysfunction associated with catecholamine resistance, which ultimately accounted for a mortality rate of 100% and a mean survival time of only twenty-nine (29) hours. The expression rate of the inducible NO synthase increased on account of the induction of sepsis. The highly selective iNOS blockade caused a reduction of the plasma NOx levels and also of the inflammatory cell infiltration into the cardiac muscle. As a result, the cardiac function was largely maintained and also the mean survival time of the wild types was about doubled. These results show that the iNOS plays a decisive role in the NO production and cardiovascular dysfunction going along with a sepsis. The inhibition of the nNOS actually led to an even higher reduction of the NOx values, surprisingly, however, not to an improved but to a reduced LV function. This manifestation was particularly pronounced in the eNOS-/- mice. Also the mean survival time of the inhibited wild-type animals increased but to a small extent and was clearly lower than that of the iNOS-inhibited animals. This shows that, on the one hand, the nNOS has a hitherto unexpected significance for the NO synthesis in the course of a sepsis and, moreover, has a cardioprotective effect. This is most likely based on the cardiomyocyte localisation of the nNOS in the sarcoplasmic reticulum and may have contributed to the sobering results of the unselective NOS inhibition as a therapeutic approach in septic shock. In all test series, the eNOS-/- animals achieved the best results. In comparison with the drug-blocked animals, these showed the lowest NOx values as well as the lowest inflammatory impairment and were therefore protected against myocardiac depression and catecholamine resistance. This was reflected also by the longest survival period with a mean survival time of 69.5 hours. The lack of increase in the iNOS expression rate in septic eNOS-/- animals conforms in relation to the iNOS with a pro-inflammatory, regulative function of the endothelial NO synthase. In survival tests, the inhibition of both the inducible and the neuronal NO synthase in eNOS-/- mice did not lead to a further increase in the mean survival time. This shows that excessive inhibition of the NO synthases associated with consecutive subphysiological NO values may have a negative effect on the survival time. The background for this may be the lack of the likewise existing positive effects of the nitrogen monoxide on the cardiac function as well as the diminished NO-mediated bactericidal effects. On the whole, the data of the present study show that NOS inhibition as a therapy for septic shock is promising only if it is highly selective, if the positive effects of nitrogen monoxide are not suppressed and if the right time for the initiation of the therapy is chosen. The fact that the central role of the endothelial NO synthase has been shown in our experiments suggests that, at least in the early phases of sepsis, a therapy by means of a selective eNOS inhibition should be attempted. For this purpose, however, further preclinical studies are required

Die Bedeutung der NO-Synthasen im murinen Modell der septischen Hämodynamik

Sepsis is one of the biggest challenges for modern medicine. The mortality rate is very high, the incidence rate is rising worldwide and the cost for the health sector is enormous. A crucial share in the pathophysiology must be attributed to cardiocirculatory failure triggered by a generalised vasodilatation as well as septic cardiomyopathy. An important mediator for these two phenomena is nitrogen monoxide. It was the object of the present dissertation to examine the nitrogen-oxide-mediated effects on septic haemodynamics in a murine CLP model and to clarify the significance attributable to the respective NO synthases in this connection. To this end, the following experiments were conducted in wild-type and eNOS-/- mice with and without a selective blockade of the inducible and neuronal NO synthase: In order to quantify the formation of nitrogen monoxide, the nitrite and nitrate levels were measured basally and after the induction of sepsis both in the plasma and in the cardiac tissue. The cardiovascular function was examined before and after the application of noradrenaline by means of invasive pressure/volume measurement via a miniature catheter. In order to measure the effect of nitrogen monoxide on the myocardial inflammatory reaction, a histological analysis of the infiltration of leucocytary cells into the cardiac muscle tissue was conducted. Likewise, the sepsis-modulated expression rate of the iNOS was measured by means of quantitative RT-PCR and in a separate population, the mean survival time of the various groups of laboratory animals was determined. Our results show that, in septic animals, there was a severe increase in the plasma NOx level as well as in the inflammatory reaction of the cardiac muscle. The consequence was a pronounced cardiovascular dysfunction associated with catecholamine resistance, which ultimately accounted for a mortality rate of 100% and a mean survival time of only twenty-nine (29) hours. The expression rate of the inducible NO synthase increased on account of the induction of sepsis. The highly selective iNOS blockade caused a reduction of the plasma NOx levels and also of the inflammatory cell infiltration into the cardiac muscle. As a result, the cardiac function was largely maintained and also the mean survival time of the wild types was about doubled. These results show that the iNOS plays a decisive role in the NO production and cardiovascular dysfunction going along with a sepsis. The inhibition of the nNOS actually led to an even higher reduction of the NOx values, surprisingly, however, not to an improved but to a reduced LV function. This manifestation was particularly pronounced in the eNOS-/- mice. Also the mean survival time of the inhibited wild-type animals increased but to a small extent and was clearly lower than that of the iNOS-inhibited animals. This shows that, on the one hand, the nNOS has a hitherto unexpected significance for the NO synthesis in the course of a sepsis and, moreover, has a cardioprotective effect. This is most likely based on the cardiomyocyte localisation of the nNOS in the sarcoplasmic reticulum and may have contributed to the sobering results of the unselective NOS inhibition as a therapeutic approach in septic shock. In all test series, the eNOS-/- animals achieved the best results. In comparison with the drug-blocked animals, these showed the lowest NOx values as well as the lowest inflammatory impairment and were therefore protected against myocardiac depression and catecholamine resistance. This was reflected also by the longest survival period with a mean survival time of 69.5 hours. The lack of increase in the iNOS expression rate in septic eNOS-/- animals conforms in relation to the iNOS with a pro-inflammatory, regulative function of the endothelial NO synthase. In survival tests, the inhibition of both the inducible and the neuronal NO synthase in eNOS-/- mice did not lead to a further increase in the mean survival time. This shows that excessive inhibition of the NO synthases associated with consecutive subphysiological NO values may have a negative effect on the survival time. The background for this may be the lack of the likewise existing positive effects of the nitrogen monoxide on the cardiac function as well as the diminished NO-mediated bactericidal effects. On the whole, the data of the present study show that NOS inhibition as a therapy for septic shock is promising only if it is highly selective, if the positive effects of nitrogen monoxide are not suppressed and if the right time for the initiation of the therapy is chosen. The fact that the central role of the endothelial NO synthase has been shown in our experiments suggests that, at least in the early phases of sepsis, a therapy by means of a selective eNOS inhibition should be attempted. For this purpose, however, further preclinical studies are required

Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes

International audienceAstrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2+ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages

HTGR Reactor Physics and Fuel Cycle Studies

The high-temperature gas-cooled reactor (HTGR) appears as a good candidate for the next generation of nuclear power plants. In the HTGR_n project of the European Union Fifth Framework Program, analyses have been performed on a number of conceptual HTGR designs, derived from reference pebble-bed and hexagonal block-type HTGR types. It is shown that several HTGR concepts are quite promising as systems for the incineration of plutonium and possibly minor actinides. These studies were mainly concerned with the investigation and intercomparison of the plutonium and actinide burning capabilities of a number of HTGR concepts and associated fuel cycles, with emphasis on the use of civil plutonium from spent LWR uranium fuel (first generation Pu) and from spent LWR MOX fuel (second generation Pu).JRC.E.4-Nuclear fuel

Circulating NOS3 Modulates Left Ventricular Remodeling following Reperfused Myocardial Infarction

<div><p>Purpose</p><p>Nitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI.</p><p>Methods</p><p>To analyze the role of circulating NOS3 we transplanted bone marrow of NOS3^−/− and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC−/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24h, 4d, 1w, 2w and 3w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori’s One-step trichrome staining.</p><p>Results</p><p>Three weeks post MI, LV end-systolic (53.2±5.9μl;***p≤0.001;n = 5) and end-diastolic volumes (82.7±5.6μl;*p<0.05;n = 5) were significantly increased in BC−/EC+, along with decreased LV developed pressure (67.5±1.8mmHg;n = 18;***p≤0.001) and increased scar size/left ventricle (19.5±1.5%;n = 13;**p≤0.01) compared to BC+/EC+ (ESV:35.6±2.2μl; EDV:69.1±2.6μl n = 8; LVDP:83.2±3.2mmHg;n = 24;scar size/LV13.8±0.7%;n = 16). Myocardial scar of BC−/EC+ was characterized by increased total collagen content (20.2±0.8%;n = 13;***p≤0.001) compared to BC+/EC+ (15.9±0.5;n = 16), and increased collagen type I and III subtypes.</p><p>Conclusion</p><p>Circulating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.</p></div

Flow chart of the presented study.

<p>In a closed chest model, animals were subjected to reperfused myocardial infarction. After 60 min of ischemia, animals were divided into two different groups: 1) 72 h post MI 2) 3 weeks post MI. Further analysis followed as depicted.</p

BC−/EC+ exhibited increased end-systolic and end-diastolic volume and decreased left ventricular function 3 weeks post MI.

<p>BC−/EC+ exhibited an increase in end-systolic (<b>A</b>) and end-diastolic volume (<b>B</b>), a significantly more pronounced decrease in stroke volume (<b>C</b>) (BC+/EC+ n = 8 and BC−/EC+ n = 5; two-way ANOVA and Bonferroni’s post hoc test or student’s t-test; * p<0.05, ** p≤ 0.01 BC+/EC+ vs. BC−/EC+; # p<0.05, ## p≤ 0.01, ### p≤ 0.001 BC+/EC+ at different time points; $p<0.05,$$p≤ 0.01,$ $$ p≤ 0.001 BC−/EC+ at different time points), and decreased left ventricular developed pressure (<b>D</b>) 3 weeks post MI compared to BC+/EC+ (BC+/EC+ n = 24 and BC−/EC+ n = 18; student‘s t-test; ***p≤0.001).</p