Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase.

The cellular response to an inflammatory stressor requires a proinflammatory cellular activation followed by a controlled resolution of the response to restore homeostasis. We hypothesized that biliverdin reductase (BVR) by binding biliverdin (BV) quells the cellular response to endotoxin-induced inflammation through phosphorylation of endothelial nitric oxide synthase (eNOS). The generated NO, in turn, nitrosylates BVR, leading to nuclear translocation where BVR binds to the Toll-like receptor-4 (TLR4) promoter at the Ap-1 sites to block transcription. We show in macrophages that BV-induced eNOS phosphorylation (Ser-1177) and NO production are mediated in part by Ca(2+)/calmodulin-dependent kinase kinase. Furthermore, we show that BVR is S-nitrosylated on one of three cysteines and that this posttranslational modification is required for BVR-mediated signaling. BV-induced nuclear translocation of BVR and inhibition of TLR4 expression is lost in macrophages derived from Enos(-/-) mice. In vivo in mice, BV provides protection from acute liver damage and is dependent on the availability of NO. Collectively, we elucidate a mechanism for BVR in regulating the inflammatory response to endotoxin that requires eNOS-derived NO and TLR4 signaling in macrophages.

Interaction of cationic ultrasmall superparamagnetic iron oxide nanoparticles with human melanoma cells.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) are currently under development for the intracellular delivery of therapeutics. However, the mechanisms of cellular uptake and the cellular reaction to this uptake, independent of therapeutics, are not well defined. The interactions of biocompatible cationic aminoUSPIONs with human cells was studied in 2D and 3D cultures using biochemical and electron microscopy techniques. AminoUSPIONs were internalized by human melanoma cells in 2D and 3D cultures. Uptake was clathrin mediated and the particles localized in lysosomes, inducing activation of the lysosomal cathepsin D and decreasing the expression of the transferrin receptor in human melanoma cells and/or skin fibroblasts. AminoUSPIONs deeply invaded 3D spheroids of human melanoma cells. Thus, aminoUSPIONs can invade tumors and their uptake by human cells induces cell reaction.

Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs.

Endothelium-derived nitric oxide (EDNO) plays a pivotal role in regulating pulmonary circulation. To determine whether there is a heterogeneity in EDNO-mediated responses of different sized pulmonary vessels, we studied small and large isolated pulmonary arteries of newborn lambs (diameter, 0.4-0.7 and 1.5-2.5 mm, respectively). The isometric tension of vessel rings were recorded while suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95% O2-5% CO2, 37 degrees C). In vessels preconstricted with norepinephrine, acetylcholine and bradykinin induced a greater relaxation of small pulmonary arteries than of large pulmonary arteries. Acetylcholine, bradykinin, and nitric oxide also induced a greater increase in cGMP content in small arteries than in large ones. The responses to acetylcholine and bradykinin were endothelium-dependent and inhibited by nitro-L-arginine, an inhibitor of nitric oxide synthase. In vessels without endothelium, the response to nitric oxide was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. The activity of soluble guanylyl cyclase of small arteries was greater than that of large arteries under basal conditions and after stimulation with S-nitroso-N-acetylpenicillamine, a nitric oxide donor. These results demonstrate that heterogeneity exists in EDNO-mediated relaxation of small and large pulmonary arteries in newborn lambs. A difference in the soluble guanylate cyclase activity of vascular smooth muscle may have contributed to this phenomenon.

Magnetic and in vitro heating properties of implants formed in situ from injectable formulations and containing superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microparticles for magnetically induced local hyperthermia

The biological and therapeutic responses to hyperthermia, when it is envisaged as an anti-tumor treatment modality, are complex and variable. Heat delivery plays a critical role and is counteracted by more or less efficient body cooling, which is largely mediated by blood flow. In the case of magnetically mediated modality, the delivery of the magnetic particles, most often superparamagnetic iron oxide nanoparticles (SPIONs), is also critically involved. We focus here on the magnetic characterization of two injectable formulations able to gel in situ and entrap silica microparticles embedding SPIONs. These formulations have previously shown suitable syringeability and intratumoral distribution in vivo. The first formulation is based on alginate, and the second on a poly(ethylene-co-vinyl alcohol) (EVAL). Here we investigated the magnetic properties and heating capacities in an alternating magnetic field (141 kHz, 12 mT) for implants with increasing concentrations of magnetic microparticles. We found that the magnetic properties of the magnetic microparticles were preserved using the formulation and in the wet implant at 37 degrees C, as in vivo. Using two orthogonal methods, a common SLP (20 Wg(-1)) was found after weighting by magnetic microparticle fraction, suggesting that both formulations are able to properly carry the magnetic microparticles in situ while preserving their magnetic properties and heating capacities. (C) 2010 Elsevier B.V. All rights reserved.

Endurance training enhances vasodilation induced by nitric oxide in human skin.

Résumé Il a été démontré que l'exercice physique modifiait le contrôle de la thermorégulation cutané, ce qui se manifeste par une augmentation de la perfusion de la microcirculation de la peau. Pour une même augmentation de température, ce phénomène est plus important chez les sportifs d'endurance que chez les sujets sédentaires. Dans cette étude, nous posons l'hypothèse qu'une composante de cette adaptation peut provenir d'une plus haute capacité des vaisseaux sanguins à répondre à un stimulus vasodilatateur. Pour la tester, nous avons recruté des hommes sains, non fumeurs, soit entraînés (surtout sport d'endurance) ou sédentaires que nous avons partagé en deux classes d'âges (18-35 ans [jeunes] et >50 ans[âgés]). Le flux sanguin cutané était mesuré par un laser-Doppler au niveau de la peau de l'avant-bras. Nous avons alors mesuré la vasodilatation obtenue par les stimuli suivant : Iontophorèse à l'acétylcholine (ACh, un vasodilatateur dépendant de l'endothélium), iontophorèse au nitroprussiate de sodium (SNP, un donneur d'oxyde nitrique) et par libération d'une interruption momentanée du flux artériel huméral (hyperémie réactive). Chez les sujets entraînés, l'effet de l'hyperémie réactive et de l'ACh n'ont pas montré de différence. Par contre, l'augmentation de la perfusion, suivant la iontophorèse de SNP, exprimé en unité de perfusion (PU), était plus importante chez les sujets entraînés que chez les sujets sédentaires (jeunes: 398±54 vs 350±87, p<0.05; âgés: 339±72 vs 307±66, p<0.05). Pour conclure, l'entraînement d'endurance augmente l'effet vasodilatateur de l'oxyde nitrique de la microcirculation cutanée humaine, au moins au niveau de la peau de l'avant-bras. Ces observations ont un intérêt physiologique considérable au vu des résultats d'études récentes qui montrent que le NO sert d'intermédiaire dans la vasodilatation cutanée produite par un stress thermique. Donc, l'augmentation de la bioactivité du NO dans la microcirculation cutanée pourrait être un des mécanismes par lequel l'entraînement physique modifierait le contrôle de la thermorégulation du flux sanguin cutané. Abstract Endurance training modifies the thermoregulatory control of skin blood flow, as manifested by a greater augmentation of skin perfusion for the same increase in core temperature in athletes, in comparison with se-dentary subjects. In this study, we tested the hypothesis that a component of this adaptation might reside in a higher ability of cutaneous blood vessels to respond to vasodilatory stimuli. We recruited healthy nonsmoking males, either endurance trained or sedentary, in two different age ranges (18-35 y and >50 y). Skin blood flow was measured in the forearm skin, using a laser Doppler imager, allowing to record the vasodilatory responses to the following stimuli: iontophoresis of acetylcholine (an endothelium-dependent vasodilator), iontophoresis of sodium nitroprusside (a nitric oxide donor), and release of a temporary interruption of arterial inflow (reactive hyperemia). There was no effect of training on reactive hyperemia or the response to acetylcholine. In contrast, the increase in perfusion following the iontophoresis of sodium nitroprusside, ex-pressed in perfusion units, was larger in trained than in sedentary subjects (younger: 398±54 vs 350±87, p<0.05; older 339±72 vs 307±66, p<0.05). In conclusion, endurance training enhances the vasodilatory effects of nitric oxide in the human dermal microcirculation, at least in forearm skin. These observations have considerable physiologic interest in view of recent data indicating that nitric oxide mediates in part the cutaneous vasodilation induced by heat stress in humans. Therefore, the augmentation of nitric oxide bioactivity in the dermal microcirculation might be one mechanism whereby endurance training modifies the thermoregulatory control of skin blood flow.

L-canavanine, an inhibitor of inducible nitric oxide synthase, improves venous return in endotoxemic rats.

OBJECTIVE: To investigate the hemodynamic effects of L-canavanine (an inhibitor of inducible, but not of constitutive, nitric oxide synthase) in endotoxic shock. DESIGN: Controlled, randomized, experimental study. SETTING: Animal laboratory. SUBJECTS: Wistar rats. INTERVENTIONS: Rats were anesthetized with pentobarbital, and hemodynamically monitored. One hour after an intravenous challenge with 5 mg/kg of Escherichia coli endotoxin, the rats were randomized to receive a continuous infusion of either L-canavanine (20 mg/kg/hr; n = 8) or vehicle only (isotonic saline, n = 11). In all animals, the infusion was given over 5 hrs at a rate of 2 mL/kg/hr. These experiments were repeated in additional rats challenged with isotonic saline instead of endotoxin (sham experiments). MEASUREMENTS AND MAIN RESULTS: Arterial blood pressure, heart rate, thermodilution cardiac output, central venous pressure, mean systemic filling pressure, urine output, arterial blood gases, blood lactate concentration, and hematocrit were measured. In sham experiments, hemodynamic stability was maintained throughout and L-canavanine had no detectable effect. Animals challenged with endotoxin and not treated with L-canavanine developed progressive hypotension and low cardiac output. After 6 hrs of endotoxemia, both central venous pressure and mean systemic filling pressure were significantly below their baseline values, indicating relative hypovolemia as the main determinant of reduced cardiac output. In endotoxemic animals treated with L-canavanine, hypotension was less marked, while cardiac output, central venous pressure, and mean systemic filling pressure were maintained throughout the experiment. L-canavanine had no effect on the time-course of hematocrit. L-canavanine significantly increased urine output and reduced the severity of lactic acidosis. CONCLUSIONS: Six hours after an endotoxin challenge in rats, low cardiac output develops, which appears to be primarily related to relative hypovolemia. L-canavanine, a selective inhibitor of the inducible nitric oxide synthase, increases the mean systemic filling pressure, thereby improving venous return, under these conditions.

Partial gene deletion of endothelial nitric oxide synthase predisposes to exaggerated high-fat diet-induced insulin resistance and arterial hypertension.

Nitric oxide (NO) plays a major role in the regulation of cardiovascular and metabolic homeostasis, as evidenced by insulin resistance and arterial hypertension in endothelial NO synthase (eNOS) null mice. Extrapolation of these findings to humans is difficult, however, because eNOS gene deficiency has not been reported. eNOS gene polymorphism and impaired NO synthesis, however, have been reported in several cardiovascular disease states and could predispose to insulin resistance. High-fat diet induces insulin resistance and arterial hypertension in normal mice. To test whether partial eNOS deficiency facilitates the development of insulin resistance and arterial hypertension during metabolic stress, we examined effects of an 8-week high-fat diet on insulin sensitivity (euglycemic clamp) and arterial pressure in eNOS(+/-) mice. When fed a normal diet, these mice had normal insulin sensitivity and were normotensive. When fed a high-fat diet, however, eNOS(+/-) mice developed exaggerated arterial hypertension and had fasting hyperinsulinemia and a 35% lower insulin-stimulated glucose utilization than control mice. The partial deletion of the eNOS gene does not alter insulin sensitivity or blood pressure in mice. When challenged with nutritional stress, however, partial eNOS deficiency facilitates the development of insulin resistance and arterial hypertension, providing further evidence for the importance of this gene in linking metabolic and cardiovascular disease.

Neuroticism modifies psychophysiological responses to fearful films

Background: Neuroticism is a personality component frequently found in anxious and depressive psychiatric disorders. The influence of neuroticism on negative emotions could be due to its action on stimuli related to fear and sadness, but this remains debated. Our goal was thus to better understand the impact of neuroticism through verbal and physiological assessment in response to stimuli inducing fear and sadness as compared to another negative emotion (disgust).¦Methods: Fifteen low neurotic and 18 high neurotic subjects were assessed on an emotional attending task by using film excerpts inducing fear, disgust, and sadness. We recorded skin conductance response (SCR) and corrugator muscle activity (frowning) as indices of emotional expression.¦Results: SCR was larger in high neurotic subjects than in low neurotics for fear relative to sadness and disgust. Moreover, corrugator activity and SCR were larger in high than in low neurotic subjects when fear was induced.¦Conclusion: After decades of evidence that individuals higher in neuroticism experience more intense emotional reactions to even minor stressors, our results indicate that they show greater SCR and expressive reactivity specifically to stimuli evoking fear rather than to those inducing sadness or disgust. Fear processing seems mainly under the influence of neuroticism. This modulation of autonomic activity by neurotics in response to threat/fear may explain their increased vulnerability to anxious psychopathologies such as PTSD (post traumatic stress disorder).

Positive contrast visualization of iron oxide-labeled stem cells using inversion-recovery with ON-resonant water suppression (IRON)

In proton magnetic resonance imaging (MRI) metallic substances lead to magnetic field distortions that often result in signal voids in the adjacent anatomic structures. Thus, metallic objects and superparamagnetic iron oxide (SPIO)-labeled cells appear as hypointense artifacts that obscure the underlying anatomy. The ability to illuminate these structures with positive contrast would enhance noninvasive MR tracking of cellular therapeutics. Therefore, an MRI methodology that selectively highlights areas of metallic objects has been developed. Inversion-recovery with ON-resonant water suppression (IRON) employs inversion of the magnetization in conjunction with a spectrally-selective on-resonant saturation prepulse. If imaging is performed after these prepulses, positive signal is obtained from off-resonant protons in close proximity to the metallic objects. The first successful use of IRON to produce positive contrast in areas of metallic spheres and SPIO-labeled stem cells in vitro and in vivo is presented.

Long-term swimming exercise does not modulate the Akt-dependent endothelial nitric oxide synthase phosphorylation in healthy mice.

Molecular mechanisms by which exercise exerts cardiovascular benefits are poorly understood. Exercise-induced increase of endothelial NO synthase (eNOS) phosphorylation through the protein kinase Akt has been shown to be a key mechanism underlying the beneficial effect of exercise in coronary artery disease patients. We examined whether this protective pathway might also be activated in long-term-exercised healthy mice. C57BL/6 wild-type mice swam for 24 weeks. A group of sedentary animals were used as controls. Aortic levels of total protein kinase Akt (protein kinase B), phosphorylated Akt at ser473 (p-Akt), total eNOS, phosphorylated eNOS at Ser1177 (p-eNOS), and PECAM-1 (platelet endothelial cell adhesion molecule-1) were assessed by Western blotting. Protein expressions of Akt, p-Akt, eNOS, p-eNOS, and PECAM-1 were not modulated by 24 weeks of exercise. The Akt-dependent eNOS phosphorylation did not seem to be a primary molecular adaptation in response to long-term exercise in healthy mice.

Regulation of catecholamine release and tyrosine hydroxylase in human adrenal chromaffin cells by interleukin-1beta: role of neuropeptide Y and nitric oxide.

Adrenal chromaffin cells synthesize and secrete catecholamines and neuropeptides that may regulate hormonal and paracrine signaling in stress and also during inflammation. The aim of our work was to study the role of the cytokine interleukin-1beta (IL-1beta) on catecholamine release and synthesis from primary cell cultures of human adrenal chromaffin cells. The effect of IL-1beta on neuropeptide Y (NPY) release and the intracellular pathways involved in catecholamine release evoked by IL-1beta and NPY were also investigated. We observed that IL-1beta increases the release of NPY, norepinephrine (NE), and epinephrine (EP) from human chromaffin cells. Moreover, the immunoneutralization of released NPY inhibits catecholamine release evoked by IL-1beta. Moreover, IL-1beta regulates catecholamine synthesis as the inhibition of tyrosine hydroxylase decreases IL-1beta-evoked catecholamine release and the cytokine induces tyrosine hydroxylase Ser40 phosphorylation. Moreover, IL-1beta induces catecholamine release by a mitogen-activated protein kinase (MAPK)-dependent mechanism, and by nitric oxide synthase activation. Furthermore, MAPK, protein kinase C (PKC), protein kinase A (PKA), and nitric oxide (NO) production are involved in catecholamine release evoked by NPY. Using human chromaffin cells, our data suggest that IL-1beta, NPY, and nitric oxide (NO) may contribute to a regulatory loop between the immune and the adrenal systems, and this is relevant in pathological conditions such as infection, trauma, stress, or in hypertension.