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Sulfur mustard (SM) is a chemical alkylating compound that primary targets lung tissue. It causes a wide variety of pathological effects in respiratory system such as chronic bronchitis, bronchiolitis obliterans, necrosis of the mucosa and inflammation, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. However, molecular and cellular mechanisms for these pathologies are still unclear. Oxidative stress (OS) induced by reactive oxygen species (ROS) is likely a significant mechanism by which SM leads to cell death and tissues injury. SM can trigger various molecular and cellular pathways that are linked to ROS generation, OS, and inflammation. Hypoxia-induced oxidative stress, reduced activity of enzymatic antioxidants, depletion of intercellular glutathione (GSH), decreased productivity of GSH-dependent antioxidants, mitochondrial dysfunction, accumulation of leukocytes and proinflammatory cytokines, and increased expression of ROS producing-related enzymes and inflammatory mediators are the major events in which SM leads to massive production of ROS and OS in pulmonary system. Therefore, understanding of these molecules and signaling pathways gives us valuable information about toxicological effects of SM on injured tissues and the way for developing a suitable clinical treatment. In this review, we aim to discuss the possible mechanisms by which SM induces excessive production of ROS, OS, and antioxidants depletion in lung tissue of exposed patients.

Enteric nervous system neuropathy causes a wide range of severe gut motility disorders. Cell replacement of lost neurons using enteric neural stem cells (ENSC) is a possible therapy for these life-limiting disorders. Here we show rescue of gut motility after ENSC transplantation in a mouse model of human enteric neuropathy, the neuronal nitric oxide synthase (nNOS -/- ) deficient mouse model, which displays slow transit in the colon. We further show that transplantation of ENSC into the colon rescues impaired colonic motility with formation of extensive networks of transplanted cells, including the development of nNOS + neurons and subsequent restoration of nitrergic responses. Moreover, post-transplantation non-cell-autonomous mechanisms restore the numbers of interstitial cells of Cajal that are reduced in the nNOS -/- colon. These results provide the first direct evidence that ENSC transplantation can modulate the enteric neuromuscular syncytium to restore function, at the organ level, in a dysmotile gastrointestinal disease model.

Background: Asthma is characterized by airflow limitation with chronic airway inflammation, hyperresponsiveness and mucus hypersecretion. NO is generated by three nitric oxide synthase (i/n/eNOSs) isoforms, but conflicting results have been reported using asthmatic mice treated with NOSs inhibitors and NOS-knockout mice. To elucidate the authentic role of NO/NOSs in asthma, we used asthmatic mice lacking all NOSs (n/i/eNOS(-/-))., Methods: Wild-type and n/i/eNOS(-/-) mice were sensitized and challenged with ovalbumin. Pathological findings and expressions of interferon (IFN)-γ, interleukin (IL)-4, -5, -10, -13 and chemokines in the lung were evaluated., Results: Decreased eosinophilic inflammation, bronchial thickening and mucus secretion, IL-4, -5 and -13, monocyte chemoattractant protein-1, eotaxin-1 and thymus and activation-regulated chemokine expressions were observed in n/i/eNOS(-/-) mice compared to wild-type, but expressions of IFN-γ and IL-10 were similar., Conclusion: Using asthmatic n/i/eNOS(-/-) mice, NO plays important roles in accelerating bronchial eosinophilic inflammation and mucus hypersecretion in the pathophysiology of asthma.

Deinococcus radiodurans (Drad) is the most radioresistant organism known. Although mechanisms that underlie the extreme radioresistance of Drad are incompletely defined, resistance to UV irradiation-induced killing was found to be greatly attenuated in an NO synthase (NOS) knockout strain of Drad (Δnos). We now show that endogenous NO production is also critical for protection of Drad against γ-irradiation (3000 Gy), a result of accelerated growth recovery, not protection against killing. NO-donor treatment rescued radiosensitization in Δnos Drad but did not influence radiosensitivity in wild type Drad. To discover molecular mechanisms by which endogenous NO confers radioresistance, metabolite profiling studies were performed. Untargeted LC-MS-based metabolite profiling in Drad quantified relative abundances of 1425 molecules and levels of 294 of these were altered by >5-fold (p < 0.01). Unexpectedly, these studies identified a dramatic perturbation in carotenoid biosynthetic intermediates in Δnos Drad, including a reciprocal switch in the pathway end-products from deoxydeinoxanthin to deinoxanthin. NO supplementation rescued these nos deletion-associated changes in carotenoid biosynthesis, and fully-restored radioresistance to wildtype levels. Because carotenoids were shown to be important contributors to radioprotection in Drad, our findings suggest that endogenously-produced NO serves to maintain a spectrum of carotenoids critical for Drad's ability to withstand radiation insult., (Copyright © 2015 Elsevier Inc. All rights reserved.)

During the neonatal period, activity-dependent neural-circuit remodelling coincides with growth and refinement of the cerebral microvasculature. Whether neural activity also influences the patterning of the vascular bed is not known. Here we show in neonatal mice, that neither reduction of sensory input through whisker trimming nor moderately increased activity by environmental enrichment affects cortical microvascular development. Unexpectedly, chronic stimulation by repetitive sounds, whisker deflection or motor activity led to a near arrest of angiogenesis in barrel, auditory and motor cortices, respectively. Chemically induced seizures also caused robust reductions in microvascular density. However, altering neural activity in adult mice did not affect the vasculature. Histological analysis and time-lapse in vivo two-photon microscopy revealed that hyperactivity did not lead to cell death or pruning of existing vessels but rather to reduced endothelial proliferation and vessel sprouting. This anti-angiogenic effect was prevented by administration of the nitric oxide synthase (NOS) inhibitor L-NAME and in mice with neuronal and inducible NOS deficiency, suggesting that excessive nitric oxide released from hyperactive interneurons and glia inhibited vessel growth. Vascular deficits persisted long after cessation of hyperstimulation, providing evidence for a critical period after which proper microvascular patterning cannot be re-established. Reduced microvascular density diminished the ability of the brain to compensate for hypoxic challenges, leading to dendritic spine loss in regions distant from capillaries. Therefore, excessive sensorimotor stimulation and repetitive neural activation during early childhood may cause lifelong deficits in microvascular reserve, which could have important consequences for brain development, function and pathology.

Objective and Design: Hepatic microvascular dysfunction is a critical event in the development of liver failure during sepsis. Activated blood cells and reactive oxygen and nitrogen species (RONS) have been implicated in the pathogenesis of sepsis., Methods: Intravital-videomicroscopy was used to determine whether RONS contribute to the recruitment of leukocytes/platelets in the hepatic microvasculature during sepsis. Six hours following cecal-ligation and puncture (CLP), disturbances of the hepatic microvasculature were assessed in WT-mice (C57Bl/6 J; n = 8), in mice lacking gp91(phox)(n = 5), overexpressing superoxide-dismutase (SOD, n = 8), in WT-mice treated with a NOS-inhibitor (L-NAME, n = 5), lacking nNOS, eNOS or iNOS (n = 5 each), treated with the NO-donor DetaNO (n = 5), in WT-mice treated with gadolinium-chloride (GdCl(2), n = 5) and compared to a group of WT-mice following a sham operation (n = 8). Six hours post-CLP, the adhesion of leukocytes and platelets in terminal hepatic venules (THV) and sinusoids was quantified., Results: In WT-mice, CLP elicited increases in the number of adherent leukocytes and platelets. Similar responses to CLP were noted in mice overexpressing SOD or lacking either eNOS or gp91(phox). The blood-cell recruitment was significantly blunted in septic iNOS-knockout mice and this response was reversed by pre-treatment with DetaNO., Conclusion: These findings suggest that iNOS-derived NO is a determinant of the pro-inflammatory phenotype assumed by the hepatic microvasculature during sepsis.

Unlabelled: Mice lacking all three nitric oxide synthase (NOS) genes remain viable even though deletion of the major downstream target of NO, soluble guanylyl cyclase, is associated with a dramatically shortened life expectancy. Moreover, findings of relatively normal flow responses in eNOS knockouts are generally attributed to compensatory mechanisms including upregulation of remaining NOS isoforms, but the alternative possibility that dietary nitrite/nitrate (NOx) may contribute to basal levels of NO signaling has never been investigated., Aim: The aim of the present study was to examine how NO signaling products (nitrosated and nitrosylated proteins) and NO metabolites (nitrite, nitrate) are affected by single NOS deletions and whether dietary NOx plays a compensatory role in any deficiency. Specifically, we sought to ascertain whether profound alterations of these products arise upon genetic deletion of either NOS isoform, inhibition of all NOS activity, NOx restriction, or all of the above., Results: Our results indicate that while some significant changes do indeed occur, they are surprisingly moderate and compartmentalized to specific tissues. Unexpectedly, even after pharmacological inhibition of all NOSs and restriction of dietary NOx intake in eNOS knockout mice significant levels of NO-related products remain. Innovation/Conclusion: These findings suggest that a yet unidentified source of NO, unrelated to NOSs or dietary NOx, may be sustaining basal NO signaling in tissues. Given the significance of NO for redox regulation in health and disease, it would seem to be important to identify the nature of this additional source of NO products as it may offer new therapeutic avenues for correcting NO deficiencies.

Nitric oxide synthase 3 (NOS3) produces nitric oxide (NO) in endothelial cells, which stimulates cyclic guanosine monophosphate (cGMP) production and thereby mediates pulmonary vasodilation. Inhibition of cGMP enzymatic cleavage by sildenafil might be involved in lung growth stimulating processes in pulmonary hypoplasia. The aim of this study was to discover insights into the transcriptional regulation of NOS3 in a mouse model of compensatory lung growth (CLG). CLG was studied in wild type animals (WT) and NOS3 knockout mice (NOS3-/-) by dry weight, DNA, and protein quantification as well as relative quantification of NOS mRNA. All assessments were done on adult female mice, 10 days after left pneumonectomy (PNX) or sham thoracotomy. Weight ratios of right NOS3-/- lungs were no different than controls. There was a compensatory increase in DNA and a noncompensating increase in protein ratios in NOS3-/- mice compared with controls. Pharmacological knockdown with the pan-NOS inhibitor l-NAME (nitro-arginine methyl ester) reduced CLG by only 8% compared with the d-NAME treated control mice. Relative quantification of lung mRNA revealed no up-regulation of NOS3 expression in WT lungs after PNX, but NOS3-/- lungs showed a 2.6-fold higher inducible NOS2 expression compared with shams. These data suggest that NOS3 loss of function alone does not impair CLG in mice, possibly because of redundancy mechanisms involving NOS2., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Endothelin-1 (ET-1) is a multifunctional peptide, which is implicated in the renal and cardiac physicology as well as in many pathologies of these systems. ET-1's actions take place after the activation of two receptors: ET(A) and ET(B). The expression of these receptors may be modulated during the pathologic process. The analysis of the distribution and level of expression of the receptors in animal models is therefore crucial., Methods: We developed a protocol for non-radioactive in situ hybridization for the mRNA of the two endothelin receptors on paraffin-embedded tissue using digoxigenin-labeled RNA probes., Results: In heart and kidney, the staining was reliable and specific. In a mouse model for endothelin/nitric oxide imbalance, cardiac ET(B) expression was reduced. The distribution of the receptors was in accordance with the actual knowledge. Differences in cell specific expression are discussed., Conclusions: We developed a protocol for the in situ hybridization of the endothelin receptors in mice. Given that the endothelin system is implicated in the development of many diseases, we believe that this protocol may be useful for a number of future preclinical studies..

Although all three nitric oxide (NO) synthases (nNOS, iNOS, and eNOS) are expressed in injured arteries, it remains to be elucidated the role of the NOSs in their entirety in the vascular lesion formation. We addressed this issue in mice deficient in all NOS genes. Vascular injury was induced by permanent ligation of a unilateral carotid artery in wild-type (WT), singly, and triply NOS(-/-) mice. Two weeks after the procedure, constrictive vascular remodeling and neointimal formation were recognized in the ligated arteries. While constrictive remodeling was noted in the nNOS(-/-) and iNOS(-/-) genotypes, it was most accelerated in the n/i/eNOS(-/-) genotype. While neointimal formation was evident in the eNOS(-/-) and nNOS(-/-) genotypes, it was also most aggravated in the n/i/eNOS(-/-) genotype. Those lesions were reversed by long-term treatment with isosorbide dinitrate, a NO donor. Finally, we examined the involvement of bone marrow-derived cells in the vascular lesion formation. Bone marrow from the WT, singly, or triply NOS(-/-) mice was transplanted into the WT mice, and then the carotid ligation was performed. Intriguingly, constrictive remodeling and neointimal formation were both similarly most exacerbated in the case of the n/i/eNOS(-/-) bone marrow transplantation. These results indicate that the complete disruption of all the NOS genes causes markedly accelerated vascular lesion formation caused by blood flow disruption in mice in vivo, demonstrating the crucial vasculoprotective role of the whole endogenous NOS system. Our findings also suggest that the NOS system in bone marrow-derived cells may be involved in this vasculoprotective mechanism., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild type (WT) and iNOS(-/-) mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides. Vaccinated WT and iNOS(-/-) mice revealed comparable fungal burden in their lungs and spleen at 7 and 11 days postchallenge. However, at 11 days the non-vaccinated, iNOS-deficient mice had significantly higher fungal burden in their spleen compared to WT mice. Additionally, higher numbers of lung-infiltrated neutrophils, macrophages and dendritic cells were observed in WT mice at day 11 postchallenge compared to iNOS(-/-) mice. Moreover, no difference in numbers of T, B, NK or regulatory T cells, or concentrations of selected cytokines and chemokines were detected in lungs of both mouse strains at 7 and 11 days postchallenge. Although iNOS-derived NO production appears to influence the inflammatory response and dissemination of the fungal pathogen, our results suggest that iNOS activity does not play a significant role in the control of coccidioidal infection in mice and that other, still undefined mechanisms of host protection are involved., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Several studies have investigated the involvement of nitric oxide (NO) in acute and chronic pain using mice lacking a single NO synthase (NOS) gene among the three isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). However, the precise role of NOS/NO in pain states remains to be determined owing to the substantial compensatory interactions among the NOS isoforms. Therefore, in this study, we used mice lacking all three NOS genes (n/i/eNOS-/-mice) and investigated the behavioral phenotypes in a series of acute and chronic pain assays., Results: In a model of tissue injury-induced pain, evoked by intraplantar injection of formalin, both iNOS-/-and n/i/eNOS-/-mice exhibited attenuations of pain behaviors in the second phase compared with that in wild-type mice. In a model of neuropathic pain, nerve injury-induced behavioral and cellular responses (tactile allodynia, spinal microglial activation and Src-family kinase phosphorylation) were reduced in n/i/eNOS-/-but not iNOS-/-mice. Tactile allodynia after nerve injury was improved by acute pharmacological inhibition of all NOSs and nNOS. Furthermore, in MG-5 cells (a microglial cell-line), interferon-γ enhanced NOSs and Mac-1 mRNA expression, and the Mac-1 mRNA increase was suppressed by L-NAME co-treatment. Conversely, the NO donor, sodium nitroprusside, markedly increased mRNA expression of Mac-1, interleukin-6, toll-like receptor 4 and P2X4 receptor., Conclusions: Our results provide evidence that the NOS/NO pathway contributes to behavioral pain responses evoked by tissue injury and nerve injury. In particular, nNOS may be important for spinal microglial activation and tactile allodynia after nerve injury.

Age-dependent renal damage is influenced by genetic background and the Fisher344xBrown Norway (F344xBN) rat is resistant to glomerular injury. In vulnerable strains, a fall in renal nitric oxide synthase (NOS) contributes to age-dependent renal damage. Here, we investigated renal NOS in young (3 months) and old (30 months) male F344xBN to test the hypothesis that renal NOS is maintained in "protected" strains. We also examined if 6 months of renin-angiotensin system (RAS) blockade using angiotensin converting enzyme inhibition (ACEI) and angiotensin receptor blockade (ARB) provides further benefit in these "protected" old rats. Aging increased tubulointerstitial injury but glomerular sclerosis was minimal and NOS and superoxide dismutase abundance increased. There was no change in the NOS inhibitor, ADMA (asymmetric dimethylarginine) or its regulatory enzymes. RAS blockade with ARB protected against tubulointerstitial injury and increased nNOSα, but ACEI, which also increased nNOSα, had no protective effect on the tubulointerstitium. We conclude that the glomerular sclerosis-resistant aged male F344xBN rat maintains renal NOS, thus reinforcing our hypothesis that progressive glomerular injury is related to renal NOS deficiency. The tubulointerstitial injury seen with aging is reversed with 6 months of ARB but not ACEI and is not associated with renal NOS., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Nitric oxide (NO) exerts a variety of biological actions under both physiological and pathological conditions. NO is synthesized by three distinct NO synthase (NOS) isoforms, including neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS), all of which are expressed in the human cardiovascular system. The roles of endogenous NO in the cardiovascular system have been investigated in pharmacological studies with NOS inhibitors and in studies with mice that lack each NOS isoform. However, in the pharmacological studies, the specificity of the NOS inhibitors continues to be an issue of debate, while in each of the NOS isoform-deficient mice, a compensatory mechanism by other NOSs that are not genetically deleted is apparently involved. Thus, the authentic roles of endogenous NO are still poorly understood. To address this issue, genetically engineered mice in which all three NOS genes are completely disrupted have been developed. In the triply n/i/eNOS(-/-) mice, but not in singly eNOS(-/-) mice, several cardiovascular phenotypes, including arteriosclerosis/atherosclerosis, myocardial infarction, and dyslipidemia, have been described. Furthermore, by using the triply NOS(-/-) mice, the roles of the NOS system in endothelium-dependent hyperpolarization and stain-induced NO production have been elucidated. These results provide novel insight into the cardiovascular role of the endogenous NO/NOS system at the molecular level. This review, based on the research outcomes obtained from the triply NOS(-/-) genetic model, summarizes the latest knowledge of the pathophysiological relevance of NO signaling in the cardiovascular system., (Copyright © 2010 Elsevier Inc. All rights reserved.)

A high protein intake is associated with increased glomerular filtration rate (GFR), which has been suggested to be mediated by reduced signaling of the tubuloglomerular feedback (TGF) mechanism. Nitric oxide (NO) has been shown to contribute to high protein-induced glomerular hyperfiltration, but the specific NO synthase (NOS) isoform responsible is not clear. In this study, a model for high-protein-induced hyperfiltration in conscious mice was developed. Using this model, we investigated the role of TGF using adenosine A(1)-receptor knockout mice lacking the TGF mechanism. Furthermore, the role of the different NOS isoforms was studied using neuronal-, inducible-, and endothelial-NOS knockout mice, and furthermore, wild-type mice acutely administered with the unspecific NOS inhibitor N(ω)-nitro-l-arginine methyl ester (100 mg/kg). GFR was measured consecutively in mice given a low-protein diet (8% casein) for 10 days, followed by a high-protein diet (50% casein) for 10 days. All mice developed high protein-induced hyperfiltration to a similar degree. These results demonstrate that high protein-induced glomerular hyperfiltration is independent of the TGF mechanism and NOS isoforms.

Aims: The precise role of the nitric oxide synthase (NOS) system in lipid metabolism remains to be elucidated. We addressed this point in mice that we have recently developed and that lack all three NOS isoforms., Methods and Results: Wild-type (WT), singly, doubly, and triply NOS(-/-) mice were fed either a regular or high-cholesterol diet for 3-5 months. The high-cholesterol diet significantly increased serum low-density lipoprotein (LDL) cholesterol levels in all the genotypes when compared with the regular diet. Importantly, when compared with the WT genotype, the serum LDL cholesterol levels in the high-cholesterol diet were significantly and markedly elevated only in the triply NOS(-/-) genotype, but not in any singly or doubly NOS(-/-) genotypes, and this was associated with remarkable atherosclerosis and sudden cardiac death, which occurred mainly in the 4-5 months after the high-cholesterol diet. Finally, hepatic LDL receptor expression was markedly reduced only in the triply NOS(-/-) genotype, accounting for the diet-induced dyslipidaemia in the genotype., Conclusion: These results provide the first direct evidence that complete disruption of all NOS genes causes severe dyslipidaemia, atherosclerosis, and sudden cardiac death in response to a high-fat diet in mice in vivo through the down-regulation of the hepatic LDL receptor, demonstrating the critical role of the whole endogenous NOS system in maintaining lipid homeostasis.

The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.

We aimed to identify which cytochrome P-450 (CYP) family/subfamily, as well as related transcription factor(s), is responsible for the estrogen-dependent synthesis of epoxyeicosatrienoic acids (EETs) to initiate shear stress-induced vasodilation. Microarray analysis indicated a significant upregulation of CYP2C29 and retinoid X receptor gamma (RXRgamma) in isolated mesenteric arteries/arterioles of female endothelial nitric oxide synthase-knockout mice, a result that was validated by real-time RT-PCR. The cannulated vessels were then perfused with 2 and 10 dyn/cm(2) shear stress, followed by collection of the perfusate to determine EET concentrations and isoforms. Shear stress dose-dependently stimulated the release of EETs into the perfusate, associated with an EET-mediated vasodilation, in which predominantly 14,15-EET and 11,12-EET contributed to the responses ( approximately 87.4% of total EETs). Transfection of vessels with CYP2C29 siRNA eliminated the release of EETs into the perfusate, which was evidenced by an abolished vasodilation, and confirmed by RT-PCR and Western blot analyses. Knockdown of RXRgamma in these vessels significantly inhibited the production of EETs, parallel to a reduced vasodilation. RXRgamma siRNA not only silenced the vascular RXRgamma expression, but synchronously downregulated CYP2C29 expression, leading to a reduced EET synthesis. In conclusion, our data provide the first evidence for a specific signaling cascade, by which estrogen potentially activates the CYP2C29 gene in the absence of nitric oxide, to synthesize EETs in response to shear stress, via an RXRgamma-related regulatory mechanism.

The role of nitric oxide (NO) derived from all three NO synthases (NOSs) in renal lesion formation remains to be fully elucidated. We addressed this point in mice lacking all NOSs. Renal injury was induced by unilateral ureteral obstruction (UUO). UUO caused significant renal lesion formation (tubular apoptosis, interstitial fibrosis, and glomerulosclerosis) in wild-type, singly, and triply NOS(-/-) mice. However, the extents of renal lesion formation were markedly and most accelerated in the triply NOS(-/-) genotype. UUO also elicited the infiltration of inflammatory macrophages, up-regulation of transforming growth factor (TGF)-β1, and induction of epithelial mesenchymal transition (EMT) in all of the genotypes; however, the extents were again largest by far in the triply NOS(-/-) genotype. Importantly, long-term treatment with the angiotensin II type 1 (AT(1))-receptor blocker olmesartan significantly prevented the exacerbation of those renal structural changes after UUO in the triply NOS(-/-) genotype, along with amelioration of the macrophage infiltration, TGF-β1 levels, and EMT. These results provide the first evidence that the complete disruption of all NOS genes results in markedly accelerated renal lesion formation in response to UUO in mice in vivo through the AT(1)-receptor pathway, demonstrating the critical renoprotective role of all NOSs-derived NO against pathological renal remodeling.

Obesity and insulin resistance are very frequently associated to the metabolic syndrome (MetS), play a pivotal role in the development of type 2 diabetes mellitus (T2DM) and increases the risk of cardiovascular disease. Although it varies among ethnic groups, the worldwide prevalence of MetS is 23% in young adults and increases with age. Remarkably, the prevalence of MetS is expected to rise during the next decades due to the acquisition of unhealthy life-style habits (sedentarism, smoking, unhealthy diet). A major pathological alteration present in the MetS is a prothrombotic state resulting from endothelial dysfunction and hypercoagulability produced by a dysbalance of coagulation factors and proteins regulating fibrinolysis. Although intensive research in recent years has identified a number of prothrombotic alterations in MetS patients, a better understanding of the molecular mechanisms underlying the relationship between MetS and atherotrombosis is required to improve prevention and treatment. In this review we discuss the main alterations in the endothelial function, coagulation cascade, fibrinolysis and platelet function promoting atherothrombosis in MetS and available mouse models exhibiting alterations in atherothrombosis.

Deinococcus radiodurans (Dr) withstands desiccation, reactive oxygen species, and doses of radiation that would be lethal to most organisms. Deletion of a gene encoding a homolog of mammalian nitric oxide synthase (NOS) severely compromises the recovery of Dr from ultraviolet (UV) radiation damage. The Deltanos defect can be complemented with recombinant NOS, rescued by exogenous nitric oxide (NO) and mimicked in the wild-type strain with an NO scavenging compound. UV radiation induces both upregulation of the nos gene and cellular NO production on similar time scales. Growth recovery does not depend on NO being present during UV irradiation, but rather can be manifested by NO addition hours after exposure. Surprisingly, nos deletion does not increase sensitivity to oxidative damage, and hydrogen peroxide does not induce nos expression. However, NOS-derived NO upregulates transcription of obgE, a gene involved in bacterial growth proliferation and stress response. Overexpression of the ObgE GTPase in the Deltanos background substantially alleviates the growth defect after radiation damage. Thus, NO acts as a signal for the transcriptional regulation of growth in D. radiodurans.

Objective: Tetrahydrobiopterin (BH(4)) deficiency is a cause of dystonia at birth. We hypothesized that BH(4) is a developmental factor determining vulnerability of the immature fetal brain to hypoxic-ischemic injury and subsequent motor deficits in newborns., Methods: Pregnant rabbits were subjected to 40-minute uterine ischemia, and fetal brains were investigated for global and focal changes in BH(4). Newborn kits were assessed by neurobehavioral tests following vehicle and sepiapterin (BH(4) analog) treatment of dams., Results: Naive fetal brains at 70% gestation (E22) were severely deficient for BH(4) compared with maternal and other fetal tissues. BH(4) concentration rapidly increased normally in the perinatal period, with the highest concentrations found in the thalamus compared with basal ganglia, frontal, occipital, hippocampus, and parietal cortex. Global sustained 40-minute hypoxia-ischemia depleted BH(4) in E22 thalamus and to a lesser extent in basal ganglia, but not in the frontal, occipital, and parietal regions. Maternal supplementation prior to hypoxia-ischemia with sepiapterin increased BH(4) in all brain regions and especially in the thalamus, but did not increase the intermediary metabolite, 7,8-BH(2). Sepiapterin treatment also reduced incidence of severe motor deficits and perinatal death following E22 hypoxia-ischemia., Interpretation: We conclude that early developmental BH(4) deficiency plays a critical role in hypoxic-ischemic brain injury. Increasing brain BH(4) via maternal supplementation may be an effective strategy in preventing motor deficits from antenatal hypoxia-ischemia.

Objective: The objective of the study was to investigate the transgenerational effect of fetal vascular programming, Study Design: Homozygous NOS3 knockout and wild type controls (NOS3+/+WT) were cross-bred to obtain heterozygous offspring that developed in (KO-/-) mothers lacking a functional NOS3 (KOM) vs wild-type control mothers (KOP). The first-generation KOM(+/-) and KOP(+/-) female mice were then bred with WT(+/+) males to obtain a second generation (F2). F2 offspring were genotyped and WT(+/+)-F2 mice were then used for in vivo blood pressure and in vitro vascular reactivity studies., Results: WT-F2 mice born to KOM mothers (KOM-F2WT) had significantly higher systolic blood pressure, mean arterial pressure, and pulse pressure, compared with WT-F2 born to KOP mothers. Male KOM-F2WT offspring had significantly increased response to phenylephrine (PE), compared with male KOP-F2WT. Male offspring had increased contractile responses to PE when compared with female. Acetylcholine responses were decreased in female KOM-F2WT, compared with female KOP-F2WT, but the difference was not statistically significant, Conclusion: Our findings support a transgenerational effect of fetal programming on the vascular phenotype and suggest possible gender specific adaptation.

The nitric oxide (NO) synthases (NOSs) system consists of three different isoforms, including neuronal (nNOS), inducible (iNOS), and endothelial NOSs (eNOS). The roles of NO in vivo have been extensively investigated in pharmacological studies with NOS inhibitors and in studies with mice lacking each NOS isoform. However, in the pharmacological studies, the specificity of NOS inhibitors continues to be an issue of debate, while in the studies with mice lacking each NOS isoform, compensatory mechanism by other NOSs appears to be involved. Thus, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. To address this important issue, we have successfully developed mice in which all three NOS genes are completely disrupted. NOS expression and activities were totally absent in the triply n/i/eNOS(-/-) mice before and after treatment with lipopolysaccharide. While the triply n/i/eNOS(-/-) mice were viable, their survival and fertility rates were markedly reduced as compared with wild-type mice. The first noticeable phenotypes were polyuria, polydipsia, and renal unresponsiveness to vasopressin, characteristics consistent with nephrogenic diabetes insipidus. We subsequently observed that in those mice, arteriosclerosis is spontaneously developed with a clustering of cardiovascular risk factors. These results provide the first evidence that genetic disruption of all three NOSs causes a variety of cardiovascular diseases in mice in vivo, demonstrating the critical role of the endogenous NOSs system in maintaining cardiovascular homeostasis.

Congenital alveolar dysplasia (CAD) is a rare disorder thought to represent alveolar growth arrest at the canalicular stage of development. An infant with CAD diagnosed on lung biopsy is reported, her respiratory problems resolved spontaneously and she was doing well at follow-up. The infant additionally suffered from systemic hypertension and hypertrophic pyloric stenosis. In conclusion, we speculate that the association of CAD with systemic hypertension and hypertrophic pyloric stenosis might be explained by abnormalities of isoforms of nitric oxide synthase (NOS) resulting in congenital deformities involving smooth muscles.

Objective: The purpose of this study was to investigate vascular reactivity in heterozygous and homozygous offspring with a genetic predisposition for hypertension after postnatal cross-fostering to mothers with the opposite genetic inheritance of the NOS3 knockout allele., Study Design: Homozygous NOS3 knockout (C57BL/6J-NOS3(-/-KO)) and wild-type mice (NOS3(+/+WT)) were bred to obtain heterozygous litters with a paternally derived (NOS3(+/-pat)) or maternally derived (NOS3(+/-mat)) knockout allele. After delivery, heterozygous and homozygous litters were cross-fostered to a mother with the opposite NOS3 gene status. Carotid arteries were placed in a wire myograph for isometric tension recording with the use of contractile and relaxant agents. Statistical analysis with 1-way analysis of variance and Neuman-Keuls post-hoc testing was performed., Results: Increased sensitivity to phenylephrine and absent relaxation to acetylcholine in NOS3(+/-mat) was reversed with cross-fostering, and vasorelaxation to isoproterenol was increased. Contraction to calcium was increased in the cross-fostered paternally derived and wild-type litters., Conclusion: Postnatal interventions may alter the adult vascular profile favorably that is the result of an abnormal intrauterine environment.

Patients with infantile hypertrophic pyloric stenosis (IHPS) have an increased incidence of renal anomalies. Association of IHPS and polycystic kidney disease (PKD) has been reported in three case histories. It is uncertain whether the association of PKD and IHPS is due to genetics, a pathophysiological link or a chance phenomenon. One hypothesis linking the two conditions is nitric oxide synthetase (NOS) deficiency. NOS deficiency leads to lack of locally available nitric oxide causing a failure of smooth muscle relaxation. It is therefore plausible that NOS deficiency plays a part in the pathogenesis of pyloric stenosis, hypertension and cyst development in autosomal-dominant PKD.

Background/aims: Early embryonic cardiomyocytes beat spontaneously. The hyperpolarization-activated cyclic-nucleotide-modulated current (I(f)) appears to be involved in its modulation as it is highly expressed at this stage. The spontaneous beating of early embryonic heart cells is slowed by acetylcholine (ACh), and our earlier studies identified a key role for nitric oxide (NO) in the regulation of the voltage dependent L-type Ca(2+) current (I(Ca,L)). The aim of the present study was to clarify whether and via which signalling pathway(s) I(f) is regulated upon muscarinic receptor activation in early embryonic (E9.5 to E11.5) cardiomyocytes., Methods: The whole-cell patch clamp technique in combination with pharmacology and/or knock out mouse models was used to investigate the regulation of I(f)., Results: We found that the ACh analogue carbachol (CCh, 10 micromol) led in the majority of cells (68%, n=50) to a significant depression of I(f) by 16.3+/-1.4% (n=34, p<0.01, voltage steps from -35 mV to -110 mV). This cholinergic inhibition was mediated by the NO/cGMP signalling pathway as it was largely reversed by superfusion with the non selective nitric oxide synthase (NOS) inhibitor N(G)-Methyl-L-arginine acetate salt (L-NMMA, 1 mmol), the inhibitor of the soluble guanylyl cyclase (sGC) 1H-[1, 2, 4]Oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ, 100 micromol) and a selective inhibitor of the phosphodiesterase (PDE) type 2 Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 micromol). Analysis of the muscarinic signalling in embryonic cardiomyocytes harvested from NOS2 (-/-) and NOS3 (-/-) mice revealed that the NOS3 isoform was entirely responsible for the muscarinic receptor-induced NO production., Conclusions: Muscarinic receptor stimulation depresses I(f) by generating NO via the NOS3 and the cGMP/PDE type 2 signalling pathway in early embryonic cardiomyocytes. This suggests that NO is a key signalling molecule involved in the regulation of chronotropy of early embryonic heart cells., (Copyright (c) 2007 S. Karger AG, Basel.)

The role of B lymphocytes in protecting the host against pulmonary Cryptococcus neoformans infection is until now, uncertain. A recent study using B lymphocyte-deficient mice suggests that B lymphocytes play a protective role in cryptococcal infection. It has been well established that at least three B cell subsets, B-1a, B-1b, and B-2, are present in the mouse periphery. B-1 cells constitute a minor fraction of the B cell population in the spleen and are not detected in lymph nodes of mice. We demonstrated that B-1 cells migrate to a nonspecific, inflammatory focus and differentiate into macrophage-like cells. However, the role these cells might play on the kinetics and evolution of the inflammatory response and on fungal infection has not yet been established. Based on these data, we decided to investigate the interaction of B-1-derived mononuclear phagocytes (BDMP) with C. neoformans to elucidate the possible influence of this cell in the progression of the disease. In this study, we demonstrated that the BDMP cell internalized C. neoformans and that this process was mediated by complement receptor 3. Thus, our results showed that the BDMP cell was more fungicidal than a macrophage and up-regulated major histocompatibility complex type II and the CD86 costimulatory molecule with the production of proinflammatory cytokines. The phagocytosis of C. neoformans results in the nitric oxide (NO)-mediated death of the fungus, as demonstrated by experiments using NO synthase 2 knockout and aminoguanidine-treated, wild-type mice.

Normal blood vessel formation is required for the development of nearly all organs during embryogenesis, including lung. Neonatal diseases such as persistent pulmonary hypertension of the newborn and alveolar capillary dysplasia are thought to result in large part from a failure of normal lung vascular development. Therefore, in the past decade, there has been increasing interest in studying the mechanisms underlying the development of the lung circulation to better understand the pathogenesis of these often lethal neonatal lung diseases. It has now been well accepted that in addition to its protean roles in the maintenance of vascular homeostasis, endothelium-derived nitric oxide also plays a pivotal role in postnatal angiogenesis, mediating downstream signaling in response to classic angiogenic factors. Recent findings in endothelial nitric oxide synthase (eNOS)-deficient mice point to a novel and previously unrecognized role of eNOS-NO pathway in fetal lung vascular development and lung morphogenesis. The lung phenotype of eNOS mutants closely resembles alveolar capillary dysplasia in humans, a universally fatal form of persistent pulmonary hypertension of the newborn that presents with defective lung vascular development and respiratory distress in newborn. We anticipate that these new insights into the basic mechanisms of lung vascular development may lead to novel therapeutic strategies for neonatal lung diseases.

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.

Basal vascular endothelial permeability is normally kept low in part by the restrictiveness of interendothelial junctions (IEJs). We investigated the possible role of nitric oxide (NO) in controlling IEJ integrity and thereby regulating basal vascular permeability. We determined the permeability of continuous endothelia in multiple murine vascular beds, including lung vasculature, of wild-type mice, endothelial nitric oxide synthase (eNOS) null mice, and mice treated with NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). Light and electron microscopic studies revealed that L-NAME treatment resulted in IEJs opening within a few minutes with a widespread response within 30 min. We observed a 35% increase in transendothelial transport of albumin, using as tracer dinitrophenylated albumin in mouse lungs and other organs studied. To rule out the involvement of blood cells in the mechanism of increased endothelial permeability, vascular beds were flushed free of blood, treated with L-NAME, and perfused with the tracer. The open IEJs observed in these studies indicated a direct role for NO in preserving the normal structure of endothelial junctions. We also used the electron-opaque tracer lanthanum chloride to assess vascular permeability. Lanthanum chloride was presented by perfusion to various vascular beds of mice lacking NO. Open IEJs were seen only in capillary and venular endothelial segments of mice lacking NO, and there was a concomitant increase in vascular permeability to the tracer. Together, these data demonstrate that constitutive eNOS-derived NO is a crucial determinant of IEJ integrity and thus serves to maintain the low basal permeability of continuous endothelia.

The genetic loss of endothelial-derived nitric oxide synthase (eNOS) in mice impairs vascular endothelial growth factor (VEGF) and ischemia-initiated blood flow recovery resulting in critical limb ischemia. This result may occur through impaired arteriogenesis, angiogenesis, or mobilization of stem and progenitor cells. Here, we show that after ischemic challenge, eNOS knockout mice [eNOS (-/-)] have defects in arteriogenesis and functional blood flow reserve after muscle stimulation and pericyte recruitment, but no impairment in endothelial progenitor cell recruitment. More importantly, the defects in blood flow recovery, clinical manifestations of ischemia, ischemic reserve capacity, and pericyte recruitment into the growing neovasculature can be rescued by local intramuscular delivery of an adenovirus encoding a constitutively active allele of eNOS, eNOS S1179D, but not a control virus. Collectively, our data suggest that endogenous eNOS-derived NO exerts direct effects in preserving blood flow, thereby promoting arteriogenesis, angiogenesis, and mural cell recruitment to immature angiogenic sprouts.

We tested the hypotheses that 1) systemic IL-10, after adenoviral gene transfer, protects arteries from impaired relaxation produced by LPS; 2) local expression of IL-10 within the arterial wall protects against vasomotor dysfunction after LPS; and 3) IL-10 protects against vascular dysfunction mediated by inducible NO synthase (iNOS) after LPS. In IL-10-deficient (IL-10-/-) and wild-type (WT, IL-10+/+) mice, LPS in vivo impaired relaxation of arteries to acetylcholine and gene transfer of IL-10 improved responses to acetylcholine. Superoxide levels were elevated in arteries after LPS, and increased levels of superoxide were prevented by gene transfer of IL-10. In arteries incubated with a low concentration of LPS in vitro to eliminate systemic effects of LPS and IL-10 from nonvascular sources, responses to acetylcholine were impaired in IL-10-deficient mice and impairment was largely prevented by gene transfer in vitro of IL-10. In arteries from WT mice in vitro, the low concentration of LPS did not impair responses to acetylcholine. Thus IL-10 within the vessel wall protects against LPS-induced dysfunction. In IL-10-deficient mice, aminoguanidine, which inhibits iNOS, protected against vasomotor dysfunction after LPS. In arteries from iNOS-deficient mice, LPS did not impair responses to acetylcholine. These findings suggest that both systemic and local effects of IL-10 provide important protection of arteries against an inflammatory stimulus and that IL-10 decreases iNOS-mediated impairment of vasorelaxation after LPS.

Pulmonary vasoconstriction in response to alveolar hypoxia (HPV) is frequently impaired in patients with sepsis or acute respiratory distress syndrome or in animal models of endotoxemia. Pulmonary vasodilation due to overproduction of nitric oxide (NO) by NO synthase 2 (NOS2) may be responsible for this impaired HPV after administration of endotoxin (LPS). We investigated the effects of acute nonspecific (N(G)-nitro-L-arginine methyl ester, L-NAME) and NOS2-specific [L-N6-(1-iminoethyl)lysine, L-NIL] NOS inhibition and congenital deficiency of NOS2 on impaired HPV during endotoxemia. The pulmonary vasoconstrictor response and pulmonary vascular pressure-flow (P-Q) relationship during normoxia and hypoxia were studied in isolated, perfused, and ventilated lungs from LPS-pretreated and untreated wild-type and NOS2-deficient mice with and without L-NAME or L-NIL added to the perfusate. Compared with lungs from untreated mice, lungs from LPS-challenged wild-type mice constricted less in response to hypoxia (69 +/- 17 vs. 3 +/- 7%, respectively, P < 0.001). Perfusion with L-NAME or L-NIL restored this blunted HPV response only in part. In contrast, LPS administration did not impair the vasoconstrictor response to hypoxia in NOS2-deficient mice. Analysis of the pulmonary vascular P-Q relationship suggested that the HPV response may consist of different components that are specifically NOS isoform modulated in untreated and LPS-treated mice. These results demonstrate in a murine model of endotoxemia that NOS2-derived NO production is critical for LPS-mediated development of impaired HPV. Furthermore, impaired HPV during endotoxemia may be at least in part mediated by mechanisms other than simply pulmonary vasodilation by NOS2-derived NO overproduction.

Nitric oxide (NO) is produced in almost all tissues and organs, exerting a variety of biological actions under physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS), including neuronal, inducible, and endothelial NOSs. Because there are substantial compensatory interactions among the NOS isoforms, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. Here, we have successfully developed mice in which all three NOS genes are completely deleted by crossbreeding singly NOS-/- mice. NOS expression and activities were totally absent in the triply NOS-/- mice before and after treatment with lipopolysaccharide. Although the triply NOS-/- mice were viable and appeared normal, their survival and fertility rates were markedly reduced as compared with the wild-type mice. Furthermore, these mice exhibited marked hypotonic polyuria, polydipsia, and renal unresponsiveness to an antidiuretic hormone, vasopressin, all of which are characteristics consistent with nephrogenic diabetes insipidus. In the kidney of the triply NOS-/- mice, vasopressin-induced cAMP production and membranous aquaporin-2 water channel expression were reduced associated with tubuloglomerular lesion formation. These results provide evidence that the NOS system plays a critical role in maintaining homeostasis, especially in the kidney.

NO has been shown to mediate angiogenesis; however, its role in vessel morphogenesis and maturation is not known. Using intravital microscopy, histological analysis, alpha-smooth muscle actin and chondroitin sulfate proteoglycan 4 staining, microsensor NO measurements, and an NO synthase (NOS) inhibitor, we found that NO mediates mural cell coverage as well as vessel branching and longitudinal extension but not the circumferential growth of blood vessels in B16 murine melanomas. NO-sensitive fluorescent probe 4,5-diaminofluorescein imaging, NOS immunostaining, and the use of NOS-deficient mice revealed that eNOS in vascular endothelial cells is the predominant source of NO and induces these effects. To further dissect the role of NO in mural cell recruitment and vascular morphogenesis, we performed a series of independent analyses. Transwell and under-agarose migration assays demonstrated that endothelial cell-derived NO induces directional migration of mural cell precursors toward endothelial cells. An in vivo tissue-engineered blood vessel model revealed that NO mediates endothelial-mural cell interaction prior to vessel perfusion and also induces recruitment of mural cells to angiogenic vessels, vessel branching, and longitudinal extension and subsequent stabilization of the vessels. These data indicate that endothelial cell-derived NO induces mural cell recruitment as well as subsequent morphogenesis and stabilization of angiogenic vessels.

Formation of NO by NO synthases (NOSs) strictly depends on tetrahydrobiopterin. Its structural analog, tetrahydro-4-aminobiopterin, is an inhibitor of all NOS isoenzymes, which prolongs allograft survival in acute murine cardiac rejection and prevents septic shock in the rat. In this study, we show that murine bone marrow-derived dendritic cells treated with tetrahydro-4-aminobiopterin had a reduced capacity to prime alloreactive murine T cells in oxidative mitogenesis. Checking for a possible influence on LPS-induced dendritic cell maturation, we found that tetrahydro-4-aminobiopterin down-regulated MHC class II expression and counteracted LPS-induced down-regulation of ICOS ligand, while expression of CD40, CD86, CD80, B7-H1, and B7-DC remained unchanged. Tetrahydro-4-aminobiopterin also reduced activation of CD4(+) T cells isolated from mice overexpressing an OVA-specific TCR by OVA-loaded murine bone marrow-derived dendritic cells, thus indicating that its effect on MHC class II expression is involved in attenuating T cell activation. In line with affecting dendritic cell function and T cell activation, tetrahydro-4-aminobiopterin impaired production of proinflammatory cytokines and the Th1 response. With regard to cell survival, tetrahydro-4-aminobiopterin induced efficient apoptosis of murine T cells but not of murine dendritic cells. Experiments with cells from inducible NOS (iNOS) knockout mice and with N(6)-(1-iminoethyl)-L-lysine, a specific inhibitor of iNOS, ruled out participation of iNOS in any of the observed effects. These findings characterize attenuation of T cell stimulatory capacity of murine bone marrow-derived dendritic cells as an immunosuppressive mechanism of tetrahydro-4-aminobiopterin that is not related to its iNOS-inhibiting properties.

Endothelial NO synthase (eNOS) expressed in the vascular endothelium or formed within platelets was postulated to inhibit platelet activation and aggregation. We have assessed the role of eNOS in platelet aggregation in vitro and in vivo by comparison of WT and eNOS-/- mice. Aggregometer studies revealed that collagen over a concentration range of 0.36-10 microg aggregated WT and eNOS-/- platelets to the same extent (10 microg: WT 86.7+/-4.7%, eNOS-/- 91+/-12%, n=6). Collagen treatment did not result in a significant increase in cGMP formation and VASP phosphorylation. Thrombin-induced P-selectin surface expression was unchanged in eNOS-/-platelets. In line with these findings no eNOS protein was detectable within the platelets of WT mice. In vivo, bleeding time after tail tip resection tended to be shorter in eNOS/- mice (WT: 116+/-35 s; eNOS-/- 109+/-37 s, n.s). Similarly, time to occlusion of the A.carotis after focal induction of thrombosis was 501+/-76 s (WT) and 457+/-95 s (eNOS-/-) (n.s.). These data demonstrate that eNOS-deficiency minimally affects platelet aggregation and is not associated with accelerated arterial thrombosis in vivo. Thus, in the mouse endothelial NO synthase does not play a major role in the autocrine modulation of platelet function and in thrombosis of conduit vessels in vivo.

The restoration of energy balance during ischemia is critical to cellular survival; however, relatively little is known concerning the regulation of neuronal metabolic pathways in response to central nervous system ischemia. AMP-activated protein kinase (AMPK), a master sensor of energy balance in peripheral tissues, is phosphorylated and activated when energy balance is low. We investigated whether AMPK might also modulate neuronal energy homeostasis during ischemia. We utilized two model systems of ischemia, middle cerebral artery occlusion in vivo and oxygen-glucose deprivation in vitro, to delineate changes in AMPK activity incurred from a metabolic stress. AMPK is highly expressed in cortical and hippocampal neurons under both normal and ischemic conditions. AMPK activity, as assessed by phosphorylation status, is increased following both middle cerebral artery occlusion and oxygen-glucose deprivation. Pharmacological inhibition of AMPK by either C75, a known modulator of neuronal ATP levels, or compound C reduced stroke damage. In contrast, activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside exacerbated damage. Mice deficient in neuronal nitric-oxide synthase demonstrated a decrease in both stroke damage and AMPK activation compared with wild type, suggesting a possible interaction between NO and AMPK activation in stroke. These data demonstrate a role for AMPK in the response of neurons during metabolic stress and suggest that in ischemia the activation of AMPK is deleterious. The ability to manipulate pharmacologically neuronal energy balance during ischemia represents an innovative approach to neuroprotection.

Estrogen has been shown to affect vascular cell and arterial function in vitro and in vivo. Here we examined the ability of estradiol (E(2)) to cause rapid arterial dilation of elastic and muscular arteries in vivo and the mechanisms involved. E(2) administration caused a rapid increase in the outer wall diameter of both types of arteries in ovariectomized female mice. This resulted from estrogen receptor (ER)-mediated stimulation of nitric oxide production, demonstrated by preinjecting the mice arteries with a soluble inhibitor of nitric oxide (monomethyl l-arginine) and by showing the absence of E(2) action in eNOS-/- mice. Rapid activation of both ERK/MAP kinase and phosphatidylinositol 3-kinase activity was found in the E(2)-exposed arteries, and inhibiting either kinase prevented the vasodilatory action of E(2). Kinase activation and vasodilator responses to E(2) were absent in either ERalpha or ERbeta knock-out mice, implicating both receptor subtypes as mediating this E(2) action. These results indicate that E(2) modulation of arterial tonus through plasma membrane ER and rapid signaling could underlie many previously observed actions of estrogen reported to occur in women.

Postsynaptic AMPA receptor (AMPAR) trafficking mediates some forms of synaptic plasticity that are modulated by NMDA receptor (NMDAR) activation and N-ethylmaleimide sensitive factor (NSF). We report that NSF is physiologically S-nitrosylated by endogenous, neuronally derived nitric oxide (NO). S-nitrosylation of NSF augments its binding to the AMPAR GluR2 subunit. Surface insertion of GluR2 in response to activation of synaptic NMDARs requires endogenous NO, acting selectively upon the binding of NSF to GluR2. Thus, AMPAR recycling elicited by NMDA neurotransmission is mediated by a cascade involving NMDA activation of neuronal NO synthase to form NO, leading to S-nitrosylation of NSF which is thereby activated, enabling it to bind to GluR2 and promote the receptor's surface expression.

The progressive rise in uterine blood flow during pregnancy is accompanied by outward hypertrophic remodeling of the uterine artery (UA). This process involves changes of the arterial smooth muscle cells and extracellular matrix. Acute increases in blood flow stimulate endothelial production of nitric oxide (NO). It remains to be established whether endothelial NO synthase (eNOS) is involved in pregnancy-related arterial remodeling. We tested the hypothesis that absence of eNOS results in a reduced remodeling capacity of the UA during pregnancy leading to a decline in neonatal outcome. UA of nonpregnant and pregnant wild-type (Nos3+/+) and eNOS-deficient (Nos3-/-) mice were collected and processed for standard morphometrical analyses. In addition, cross sections of UA were processed for cytological (smoothelin, smooth muscle alpha-actin) and proliferation (Ki-67) immunostaining. We compared the pregnancy-related changes longitudinally and, together with the data on pregnancy outcome, transversally by analysis of variance with Bonferroni correction. During pregnancy, the increases in radius and medial cross sectional area of Nos3-/- UA was significantly less than those of Nos3+/+ UA. Smooth muscle cell dedifferentiation and proliferation were impaired in gravid Nos3-/- mice as deduced from the lack of change in the expression of smoothelin and smooth muscle alpha-actin, and the reduced Ki-67 expression. Until 17 days of gestation, litter size did not differ between both genotypes, but at birth the number of viable newborn pups and their weights were smaller in Nos3-/- than in Nos3+/+ mice. We conclude that absence of eNOS adversely affects UA remodeling in pregnancy, which may explain the impaired pregnancy outcome observed in these mice.

In visceral leishmaniasis, chemotherapy probably seldom eradicates all parasites in tissue macrophages; nevertheless, most T cell-intact patients show long-lasting clinical cure after treatment despite residual intracellular infection. To characterize prevention of posttreatment relapse, amphotericin B was used to kill approximately 90-95% of Leishmania donovani in livers of mice deficient in mechanisms of acquired antileishmanial resistance. Recrudescence subsequently developed 1) in animals deficient in both CD4 and CD8 T cells as well as CD40L-mediated T cell costimulation, but not in a) CD4 or CD8 cells alone, b) NK cell lytic activity, or c) ICAM-1-recruited monocytes; and 2) in mice deficient in IFN-gamma, but not in the IFN-gamma-inducing cytokines, a) IL-12, b) IL-12 and IL-23, or c) IL-18. Posttreatment recrudescence also did not develop in animals deficient in macrophage phagocyte NADPH oxidase (phox) or inducible NO synthase (iNOS) alone or, surprisingly, in those deficient in both phox and iNOS. Therefore, regulation of the intracellular replication of residual Leishmania donovani that escape chemotherapy evolves to a host mechanism distinguishable from initial acquired resistance at the T cell, cytokine, and macrophage levels. Posttreatment, either CD8 or CD4 cells can direct the response, IL-12 is not required, and iNOS and phox, the activated macrophage's primary IFN-gamma-inducible leishmanicidal pathways, both become dispensable.

The injection of repeated doses of lipopolysaccharide (LPS) results in attenuation of the febrile response, which is called endotoxin tolerance. We tested the hypothesis that nitric oxide (NO) arising from inducible NO synthase (iNOS) plays a role in endotoxin tolerance, using not only pharmacological trials but also genetically engineered mice. Body core temperature was measured by biotelemetry in mice treated with NG-monomethyl-L-arginine (L-NMMA, 40 mg/kg; a nonselective NO synthase inhibitor) or aminoguanidine (AG, 10 mg/kg; a selective iNOS inhibitor) and in mice deficient in the iNOS gene (iNOS KO) mice. Tolerance to LPS was induced by means of three consecutive LPS (100 microg/kg) intraperitoneal injections at 24-h intervals. In wild-type mice, we observed a significant reduction of the febrile response to repeated administration of LPS. Injection of L-NMMA and AG markedly enhanced the febrile response to LPS in tolerant animals. Conversely, iNOS-KO mice repeatedly injected with LPS did not become tolerant to the pyrogenic effect of LPS. These data are consistent with the notion that NO modulates LPS tolerance in mice and that iNOS isoform is involved in NO synthesis during LPS tolerance.

The balance between thrombosis and hemorrhage is carefully regulated. Nitric oxide (NO) is an important mediator of these processes, as it prevents platelet adhesion to the endothelium and inhibits platelet recruitment. Although endothelial NO synthase (eNOS)-deficient mice have decreased vascular reactivity and mild hypertension, enhanced thrombosis in vivo has not been demonstrated. To determine the role of endogenous NO in hemostasis, a model of carotid arterial injury and thrombosis was performed using eNOS-deficient and wild-type mice. Paradoxically, the eNOS-deficient animals had a prolongation of time to occlusion compared with the wild-type mice (P < 0.001). Consistent with this finding, plasma markers suggesting enhanced fibrinolysis [tissue plasminogen activator (t-PA) activity and antigen and D-dimer levels] were significantly elevated in eNOS-deficient animals. Vascular tissue expression of t-PA and platelet activity levels were not altered. In endothelial cells, t-PA is stored in Weibel-Palade bodies, and exocytosis of these storage granules is inhibited by NO. Thus in the absence of NO, release of Weibel-Palade body contents (and t-PA) could be enhanced; this observation is also supported by increased von Willebrand factor levels observed in eNOS-deficient animals. In summary, although eNOS deficiency attenuates vascular reactivity and increases platelet recruitment, it is also associated with enhanced fibrinolysis due to lack of NO-dependent inhibition of Weibel-Palade body release. These processes highlight the complexity of NO-dependent regulation of vascular homeostasis. Such compensatory mechanisms may partially explain the lack of spontaneous thrombosis, minimally elevated baseline blood pressure, and normal life span that are seen in animals deficient in a pivotal regulator of vascular patency.

Objective: To investigate whether stimulation with lipopolysaccharide (LPS) alters cytokine production by splenocytes in mice and whether it changes the T-helper 1 (Th1)/Th2 balance. The role of nitric oxide in such immunologic changes was also explored using mice with genetic lack of inducible nitric oxide synthase (iNOS)., Design: : Prospective animal study with concurrent controls., Setting: University research laboratory., Subjects: iNOS knockout mice and wild-type littermates., Interventions: iNOS knockout mice or wild-type mice were injected with LPS or saline with or without anti-interleukin (IL)-6 antibody, and survival was monitored for 7 days., Measurements and Main Results: At 24 and 48 hrs after administration, blood samples and splenocytes were obtained to examine immunologic variables. Cell surface markers and cytokine expression of splenocytes were used to characterize the Th1/Th2 balance and were measured by flow cytometry. At 48 hrs after LPS administration, the Th1/Th2 balance shifted toward Th2 predominance in wild-type mice, irrespective of the IL-6 level, whereas it showed Th1 predominance in iNOS knockout mice, and the increase of IL-6 and IL-10 in response to LPS persisted in these animals. After LPS administration, the mortality rate was significantly higher in iNOS knockout mice than in wild-type mice, irrespective of the IL-6 level., Conclusions: These findings suggest that nitric oxide produced by iNOS during endotoxemia may be involved in down-regulation of Th1 cytokines and up-regulation of Th2 cytokines, whereas IL-6 has no such role. The increased lethality of LPS in iNOS knockout mice suggests that nitric oxide may be protective against proinflammatory cytokine-induced damage. Nitric oxide excess may increase susceptibility to nosocomial infections, so-called immunoparalysis.

The balance of arginine metabolism via nitric oxide synthase (NOS) or arginase is an important determinant of the inflammatory response of murine macrophages and dendritic cells. Here we analyzed the expression of the isoform arginase I in human myeloid cells. Using healthy donors and patients with arginase I deficiency, we found that in human leukocytes arginase I is constitutively expressed only in granulocytes and is not modulated by a variety of proinflammatory and anti-inflammatory stimuli in vitro. We demonstrate that arginase I is localized in azurophil granules of neutrophils and constitutes a novel antimicrobial effector pathway, likely through arginine depletion in the phagolysosome. Our findings demonstrate important differences between murine and human leukocytes with respect to regulation and function of arginine metabolism via arginase.

The effects of neuronal, endothelial, or inducible nitric oxide synthase gene disruption on the expression of oxytocin and vasopressin gene were examined in the hypothalamus (paraventricular, supraoptic, suprachiasmatic, and anterior commissural nuclei) and extrahypothalamus (bed nucleus of the stria terminalis). The oxytocin messenger RNA levels in the anterior commissural nucleus of neuronal nitric oxide synthase knockout mice were significantly higher than in control mice, but not in endothelial or inducible nitric oxide synthase knockout mice. In contrast, no significant effects of neuronal, endothelial, or inducible nitric oxide synthase gene disruption on oxytocin and vasopressin messenger RNA levels in the other hypothalamic and extrahypothalamic nuclei were observed. These results suggest that neuronal nitric-oxide-synthase-derived nitric oxide may be involved in the regulation of oxytocin gene expression in the anterior commissural nucleus.