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1.  eNOS phosphorylation on serine 1176 affects insulin sensitivity and adiposity 
Phosphorylation of endothelial nitric oxide synthase (eNOS) is an important regulator of its enzymatic activity. We generated knockin mice expressing phosphomimetic (SD) and unphosphorylatable (SA) eNOS mutations at S1176 to study the role of eNOS phosphorylation. The single amino acid SA mutation is associated with hypertension and decreased vascular reactivity, while the SD mutation results in increased basal and stimulated endothelial NO production. In addition to these vascular effects, modulation of the S1176 phosphorylation site resulted in unanticipated effects on metabolism. The eNOS SA mutation results in insulin resistance, hyperinsulinemia, adiposity, and increased weight gain on high fat. In contrast, the eNOS SD mutation is associated with decreased insulin levels and resistance to high fat-induced weight gain. These results demonstrate the importance of eNOS in regulation of insulin sensitivity, energy metabolism, and bodyweight regulation, and suggest eNOS phosphorylation as a novel target for the treatment of obesity and insulin resistance.
doi:10.1016/j.bbrc.2012.12.110
PMCID: PMC3576142  PMID: 23291238
nitric oxide; endothelium; insulin resistance; diabetes; metabolism; obesity
2.  Near-infrared fluorescent imaging of matrix metalloproteinase activity following myocardial infarction 
Circulation  2005;111(14):1800-1805.
We used a molecular probe activated by protease cleavage to image expression of matrix metalloproteinases (MMPs) in the heart following myocardial infarction.
Methods and Results
We synthesized and characterized a near-infrared fluorescent (NIRF) probe that is activated by proteolytic cleavage by MMP2 and MMP9. The NIRF probe was injected into mice at various time-points up to 4 weeks after myocardial infarction induced by ligation of the left anterior descending artery. NIRF imaging of MMP activity increased in the infarct region with maximal expression at one to two weeks, persisting to four weeks. Zymography and real-time PCR analysis showed that MMP9 expression is increased at two to four days, while MMP2 expression is increased at one to two weeks. Dual label confocal microscopy showed colocalization of NIRF imaging with neutrophils on day 2, and flow cytometric analysis confirmed that NIRF signal is associated with leukocytes in the infarct zone.
Conclusion
This study demonstrates that the activity of MMPs in the myocardium may be imaged using specific activity-dependent molecular probes.
doi:10.1161/01.CIR.0000160936.91849.9F
PMCID: PMC3733536  PMID: 15809374
matrix metalloproteinases; Myocardial infarction; Imaging; Gelatinase; Remodelling
3.  Cerebral blood volume affects blood–brain barrier integrity in an acute transient stroke model 
Insufficient vascular reserve after an ischemic stroke may induce biochemical cascades that subsequently deteriorate the blood–brain barrier (BBB) function. However, the direct relationship between poor cerebral blood volume (CBV) restoration and BBB disruption has not been examined in acute stroke. To quantify BBB integrity at acute stages of transient stroke, in particular for cases in which extravasation of the standard contrast agent (Gd-DTPA) is not observed, we adopted the water exchange index (WEI), a novel magnetic resonance image-derived parameter to estimate the water permeability across the BBB. The apparent diffusion coefficient (ADC) and R2 relaxation rate constant were also measured for outlining the tissue abnormality, while fractional CBV and WEI were quantified for assessing vascular alterations. The significantly decreased ADC and R2 in the ischemic cortices did not correlate with the changes in CBV or WEI. In contrast, a strong negative correlation between the ipsilesional WEI and CBV was found, in which stroke mice were clustered into two groups: (1) high WEI and low CBV and (2) normal WEI and CBV. The low CBV observed for mice with a disrupted BBB, characterized by a high WEI, indicates the importance of CBV restoration for maintaining BBB stability in acute stroke.
doi:10.1038/jcbfm.2013.27
PMCID: PMC3677109  PMID: 23462571
blood–brain barrier; cerebral blood volume; stroke; water exchange; MRI
4.  Hyperlipidemia disrupts cerebrovascular reflexes and worsens ischemic perfusion defect 
Hyperlipidemia is a highly prevalent risk factor for coronary and cervical atherosclerosis and stroke. However, even in the absence of overt atherosclerosis, hyperlipidemia disrupts endothelial and smooth muscle function. We investigated the impact of hyperlipidemia on resting-brain perfusion, fundamental cerebrovascular reflexes, and dynamic perfusion defect during acute focal ischemia in hyperlipidemic apolipoprotein E knockout mice before the development of flow-limiting atherosclerotic stenoses. Despite elevated blood pressures, absolute resting cerebral blood flow was reduced by 20% in apolipoprotein E knockout compared with wild type when measured by [14C]-iodoamphetamine technique. Noninvasive, high spatiotemporal resolution laser speckle flow imaging revealed that the lower autoregulatory limit was elevated in apolipoprotein E knockout mice (60 vs. 40 mm Hg), and cortical hyperemic responses to hypercapnia and functional activation were attenuated by 30% and 64%, respectively. Distal middle cerebral artery occlusion caused significantly larger perfusion defects and infarct volumes in apolipoprotein E knockout compared with wild type. Cerebrovascular dysfunction showed a direct relationship to the duration of high-fat diet. These data suggest that hyperlipidemia disrupts cerebral blood flow regulation and diminishes collateral perfusion in acute stroke in the absence of hemodynamically significant atherosclerosis.
doi:10.1038/jcbfm.2013.38
PMCID: PMC3677117  PMID: 23486293
apolipoprotein E knockout mice; autoregulation; hypercapnia; hypercholesterolemia; middle cerebral artery occlusion; neurovascular coupling
5.  C-Reactive Protein Causes Insulin Resistance in Mice Through Fcγ Receptor IIB–Mediated Inhibition of Skeletal Muscle Glucose Delivery 
Diabetes  2013;62(3):721-731.
Elevations in C-reactive protein (CRP) are associated with an increased risk of insulin resistance. Whether CRP plays a causal role is unknown. Here we show that CRP transgenic mice and wild-type mice administered recombinant CRP are insulin resistant. Mice lacking the inhibitory Fcγ receptor IIB (FcγRIIB) are protected from CRP-induced insulin resistance, and immunohistochemistry reveals that FcγRIIB is expressed in skeletal muscle microvascular endothelium and is absent in skeletal muscle myocytes, adipocytes, and hepatocytes. The primary mechanism in glucose homeostasis disrupted by CRP is skeletal muscle glucose delivery, and CRP attenuates insulin-induced skeletal muscle blood flow. CRP does not impair skeletal muscle glucose delivery in FcγRIIB−/− mice or in endothelial nitric oxide synthase knock-in mice with phosphomimetic modification of Ser1176, which is normally phosphorylated by insulin signaling to stimulate nitric oxide–mediated skeletal muscle blood flow and glucose delivery and is dephosphorylated by CRP/FcγRIIB. Thus, CRP causes insulin resistance in mice through FcγRIIB-mediated inhibition of skeletal muscle glucose delivery.
doi:10.2337/db12-0133
PMCID: PMC3581204  PMID: 23069625
6.  Phosphomimetic Modulation of eNOS Improves Myocardial Reperfusion and Mimics Cardiac Postconditioning in Mice 
PLoS ONE  2014;9(1):e85946.
Objective
Myocardial infarction resulting from ischemia-reperfusion injury can be reduced by cardiac postconditioning, in which blood flow is restored intermittently prior to full reperfusion. Although key molecular mechanisms and prosurvival pathways involved in postconditioning have been identified, a direct role for eNOS-derived NO in improving regional myocardial perfusion has not been shown. The objective of this study is to measure, with high temporal and spatial resolution, regional myocardial perfusion during ischemia-reperfusion and postconditioning, in order to determine the contribution of regional blood flow effects of NO to infarct size and protection.
Methods and Results
We used myocardial contrast echocardiography to measure regional myocardial blood flow in mice over time. Reperfusion after myocardial ischemia-reperfusion injury is improved by postconditioning, as well as by phosphomimetic eNOS modulation. Knock-in mice expressing a phosphomimetic S1176D form of eNOS showed improved myocardial reperfusion and significantly reduced infarct size. eNOS knock-out mice failed to show cardioprotection from postconditioning. The size of the no-reflow zone following ischemia-reperfusion is substantially reduced by postconditioning and by the phosphomimetic eNOS mutation.
Conclusions and Significance
Using myocardial contrast echocardiography, we show that temporal dynamics of regional myocardial perfusion restoration contribute to reduced infarct size after postconditioning. eNOS has direct effects on myocardial blood flow following ischemia-reperfusion, with reduction in the size of the no-reflow zone. These results have important implications for ongoing clinical trials on cardioprotection, because the degree of protective benefit may be significantly influenced by the regional hemodynamic effects of eNOS-derived NO.
doi:10.1371/journal.pone.0085946
PMCID: PMC3897570  PMID: 24465805
7.  Atheroprotective effects of neuronal nitric oxide synthase in apolipoprotein-E knockout mice 
Arteriosclerosis, thrombosis, and vascular biology  2006;26(7):10.1161/01.ATV.0000223143.88128.19.
Background
All three isoforms of the nitric oxide synthase (NOS) are expressed in the atherosclerotic plaque. To test whether neuronal NOS (nNOS) deficiency affects atherosclerosis, we studied “western-type” diet fed apoE/nNOSα double knockout (DKO) mice, which carry a deletion of nNOS exon 2 and apoE knockout (KO) control animals.
Methods and Results
Mice were fed a “western type” diet for 14 or 24 weeks. After 14 weeks male DKO animals showed a significant 66% increase in lesion-area. Lesion-area in female DKO animals was unchanged at 14 weeks, but significantly increased by 31% after 24 weeks. Moreover, mean arterial blood pressure was significantly reduced in female DKO animals, but was unchanged in male animals. Immunohistochemistry revealed strong nNOS expression in the neointima of KO mice. In DKO animals residual nNOS staining was due to the presence of nNOS splice variants. While nNOSα was present in vessels of KO and absent in DKO animals, nNOSγ was expressed in KO and DKO mice.
Conclusion
nNOSα protects against atherosclerosis as nNOSα deletion leads to an increase in plaque formation in apoE/nNOSα DKO mice. Female DKO mice showed a significant reduction in mean arterial blood pressure which was not observed in male animals. Additionally, we found expression of nNOS splice variants in vessels of apoE KO mice. Our data highlights nNOSα overexpression as a potential therapeutic strategy and naturally occurring splice variants that lack exon 2 of the nNOS gene as a potential risk factor for vascular disease.
doi:10.1161/01.ATV.0000223143.88128.19
PMCID: PMC3845660  PMID: 16627802
8.  Reduction of Hippocampal Cell Death and Proteolytic Responses in Tissue Plasminogen Activator Knockout Mice after Transient Global Cerebral Ischemia 
Neuroscience  2007;150(1):10.1016/j.neuroscience.2007.06.029.
Knockout mice deficient in tissue plasminogen activator (tPA) are protected against hippocampal excitotoxicity. But it is unknown whether similar neuroprotection occurs after transient global cerebral ischemia, which is known to selectively affect the hippocampus. In this study, we tested the hypothesis that hippocampal cell death in tPA knockout mice would be reduced after transient global cerebral ischemia, and this neuroprotection would occur concomitantly with amelioration of both intra- and extra-cellular proteolytic cascades. Wildtype and tPA knockout mice were subjected to 20 min of transient bilateral occlusions of the common carotid arteries. Three days later, Nissl and TUNEL staining demonstrated that hippocampal cell death was significantly reduced in tPA knockout brains compared to wildtype brains. Caspase-3 and the two major brain gelatinases (MMP-9 and MMP-2) were assessed as representative measurements of intra- and extra-cellular proteolysis. Post-ischemic levels of caspase-3, MMP-9 and MMP-2 were similarly reduced in tPA knockouts compared with wildtype hippocampi. Taken together, these data suggest that endogenous tPA contributes to hippocampal injury after cerebral ischemia, and these pathophysiologic pathways may involve links to aberrant activation of caspases and MMPs.
doi:10.1016/j.neuroscience.2007.06.029
PMCID: PMC3814181  PMID: 17936515
excitotoxicity; anoikis; neuron; caspase; matrix metalloproteinase
9.  Anti-Inflammatory Effect of Targeted Delivery of SOD to Endothelium: Mechanism, Synergism with NO Donors and Protective Effects In Vitro and In Vivo 
PLoS ONE  2013;8(10):e77002.
Pro-inflammatory activation of vascular endothelium is implicated in pathogenesis of severe conditions including stroke, infarction and sepsis. We have recently reported that superoxide dismutase (SOD) conjugated with antibodies (Ab/SOD) that provide targeted delivery into endothelial endosomes mitigates inflammatory endothelial activation by cytokines and agonists of Toll-like receptors (TLR). The goal of this study was to appraise potential utility and define the mechanism of this effect. Ab/SOD, but not non-targeted SOD injected in mice alleviated endotoxin-induced leukocyte adhesion in the cerebral vasculature and protected brain from ischemia-reperfusion injury. Transfection of endothelial cells with SOD, but not catalase inhibited NFκB signaling and expression of Vascular Cell Adhesion Molecule-1 induced by both cytokines and TLR agonists. These results affirmed that Ab/SOD-quenched superoxide anion produced by endothelial cells in response to proinflammatory agents mediates NFκB activation. Furthermore, Ab/SOD potentiates anti-inflammatory effect of NO donors in endothelial cells in vitro, as well as in the endotoxin-challenged mice. These results demonstrate the central role of intracellular superoxide as a mediator of pro-inflammatory activation of endothelium and support the notion of utility of targeted interception of this signaling pathway for management of acute vascular inflammation.
doi:10.1371/journal.pone.0077002
PMCID: PMC3795626  PMID: 24146950
10.  Rhesus macaques develop metabolic syndrome with reversible vascular dysfunction responsive to pioglitazone 
Circulation  2011;124(1):77-86.
Background
The metabolic syndrome (MetS) is a constellation of clinical features that include central obesity, hypertension, atherogenic dyslipidemia, and insulin resistance (IR). However, the concept remains controversial; it has been debated whether MetS represents nothing more than simultaneous co-occurrence of individual risk factors, or whether there are common, shared pathophysiologic mechanisms that link the individual components.
Methods and Results
To investigate the emergence of metabolic and cardiovascular components during the development of MetS, we identified MetS-predisposed animals (n=35) in a large population of rhesus macaques (Macaca mulatta, 12.7 ± 2.9 years old, n=408), acclimated them to standardized conditions, and monitored the progression of individual component features over 18 months. In total 18 MetS animals with recently developed fasting hyperinsulinemia, central obesity, hypertension, and atherogenic dyslipidemia, we found that individual metabolic and cardiovascular components track together during the transition from pre-MetS to onset of MetS; MetS was associated with a 60% impairment of flow mediated dilation (FMD), establishing the mechanistic link with vascular dysfunction. Pioglitazone treatment (3 mg/kg body weight/day for 6 weeks), a PPARγ agonist, reversibly improved atherogenic dyslipidemia and IR, and fully restored FMD with persistent benefits.
Conclusions
Co-emergence of metabolic and cardiovascular components during MetS progression and complete normalization of vascular dysfunction with PPARγ agonists suggest shared underlying mechanisms rather than separate processes, arguing for the benefit of early intervention of MetS components. Predictive NHP models of MetS should be highly valuable in mechanistic and translational studies on the pathogenesis of MetS in relation to cardiovascular disease and diabetes.
doi:10.1161/CIRCULATIONAHA.110.990333
PMCID: PMC3775509  PMID: 21690491
Metabolic Syndrome (MetS); Nonhuman Primates; Cardiovascular Disease; Pathogenesis; PPARγ agonists
11.  Effect of Blood Nitrite and Nitrate Levels on Murine Platelet Function 
PLoS ONE  2013;8(2):e55699.
Nitric oxide (NO) appears to play an important role in the regulation of thrombosis and hemostasis by inhibiting platelet function. The discovery of NO generation by reduction of nitrite (NO2−) and nitrate (NO3−) in mammals has led to increased attention to these anions with respect to potential beneficial effects in cardiovascular diseases. We have previously shown that nitrite anions at 0.1 µM inhibit aggregation and activation of human platelet preparations in vitro in the presence of red blood cells and this effect was enhanced by deoxygenation, an effect likely due to NO generation. In the present study, we hypothesized that nitrite and nitrate derived from the diet could also alter platelet function upon their conversion to NO in vivo. To manipulate the levels of nitrite and nitrate in mouse blood, we used antibiotics, NOS inhibitors, low nitrite/nitrate (NOx) diets, endothelial NOS knock-out mice and also supplementation with high levels of nitrite or nitrate in the drinking water. We found that all of these perturbations affected nitrite and nitrate levels but that the lowest whole blood values were obtained by dietary restriction. Platelet aggregation and ATP release were measured in whole blood and the results show an inverse correlation between nitrite/nitrate levels and platelet activity in aggregation and ATP release. Furthermore, we demonstrated that nitrite-supplemented group has a prolonged bleeding time compared with control or low NOx diet group. These results show that diet restriction contributes greatly to blood nitrite and nitrate levels and that platelet reactivity can be significantly affected by these manipulations. Our study suggests that endogenous levels of nitrite and nitrate may be used as a biomarker for predicting platelet function and that dietary manipulation may affect thrombotic processes.
doi:10.1371/journal.pone.0055699
PMCID: PMC3562242  PMID: 23383344
12.  The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo 
Journal of Clinical Investigation  2007;117(7):1961-1967.
NO plays critical roles in vascular function. We show that modulation of the eNOS serine 1179 (S1179) phosphorylation site affects vascular reactivity and determines stroke size in vivo. Transgenic mice expressing only a phosphomimetic (S1179D) form of eNOS show greater vascular reactivity, develop less severe strokes, and have improved cerebral blood flow in a middle cerebral artery occlusion model than mice expressing an unphosphorylatable (S1179A) form. These results provide a molecular mechanism by which multiple diverse cardiovascular risks, such as diabetes and obesity, may be centrally integrated by eNOS phosphorylation in vivo to influence blood flow and cardiovascular disease. They also demonstrate the in vivo relevance of posttranslational modification of eNOS in vascular function.
doi:10.1172/JCI29877
PMCID: PMC1884686  PMID: 17557122
13.  Role of endothelial nitric oxide in cerebrovascular regulation 
Current Pharmaceutical Biotechnology  2011;12(9):1334-1342.
Endothelial nitric oxide (NO) plays important roles in the vascular system. Animal models that show vascular dysfunction demonstrate the protective role of endothelial NO dependent pathways. This review focuses on the role of endothelial NO in the regulation of cerebral blood flow and vascular tone. We will discuss the importance of NO in cerebrovascular function using animal models with altered endothelial NO production under normal, ischemic and reperfusion conditions, as well as in hyperoxia. Pharmacological and genetic manipulations of the endothelial NO system demonstrate the essential roles of endothelial NO synthase in maintenance of vascular tone and cerebral perfusion under normal and pathological conditions.
PMCID: PMC3196792  PMID: 21235451
cerebrovascular regulation; endothelial nitric oxide synthase; mutant mice
14.  Aldosterone Regulates Vascular Gene Transcription via Oxidative Stress-Dependent And –Independent Pathways 
Objective
Aldosterone (Aldo) antagonism prevents cardiovascular mortality by unclear mechanisms. Aldo binds to the mineralocorticoid receptor (MR), a ligand-activated transcription factor, which is expressed in human vascular cells. Here we define the early Aldo-regulated vascular transcriptome and investigate the mechanisms of gene regulation by Aldo in the vasculature that may contribute to vascular disease.
Methods and Results
Gene expression profiling of Aldo-treated mouse aortas identified 72 genes regulated by Aldo. These genes are overrepresented in Gene Ontology categories involved in vascular function and disease. QRT-PCR was used to confirm and further explore mechanisms of vascular gene regulation by Aldo. Aldo-regulated vascular gene expression was inhibited by actinomycin-D and MR antagonists supporting a transcriptional MR-dependent mechanism. Aldo regulation of a subset of genes was enhanced in the setting of vascular endothelial denudation and blocked by the free radical scavenger Tempol, supporting synergy between Aldo and vascular injury that is oxidative stress-dependent. In the aortic arch, a region predisposed to atherosclerosis, the injury-enhanced genes also demonstrated enhanced expression compared to the descending aorta, both at baseline and after Aldo exposure. Furthermore, the clinically beneficial MR antagonist spironolactone inhibited expression of the identified genes in aortic tissue from humans with atherosclerosis.
Conclusions
This study defines the Aldo-regulated vascular transcriptome and characterizes a subset of proatherogenic genes with enhanced Aldo-stimulated, oxidative stress-dependent expression in the setting of vascular injury and in areas predisposed to atherosclerosis. Inhibition of MR regulation of these genes may play a role in the protective effects of Aldo antagonists in patients with vascular disease and these pathways may provide novel drug targets to prevent atherosclerosis in humans.
doi:10.1161/ATVBAHA.111.229070
PMCID: PMC3141105  PMID: 21617142
15.  Delayed paraplegia after spinal cord Ischemic injury requires caspase-3 activation in mice 
Background and purpose
Delayed paraplegia remains a devastating complication after ischemic spinal cord injury associated with aortic surgery and trauma. While apoptosis has been implicated in the pathogenesis of delayed neurodegeneration, mechanisms responsible for the delayed paraplegia remain incompletely understood. The aim of this study was to elucidate the role of apoptosis in delayed motor neuron degeneration after spinal cord ischemia.
Methods
Mice were subjected to spinal cord ischemia induced by occlusion of the aortic arch and left subclavian artery for 5 or 9 min. Motor function in the hind limb was evaluated up to 72h after spinal cord ischemia. Histological studies were performed to detect caspase-3 activation, glial activation, and motor neuron survival in the serial spinal cord sections. To investigate the impact of caspase-3 activation on spinal cord ischemia, outcome of the spinal cord ischemia was examined in mice deficient for caspase-3.
Results
In wild-type mice, 9 min of spinal cord ischemia caused immediate paraplegia, whereas 5 min of ischemia caused delayed paraplegia. Delayed paraplegia after 5 min of spinal cord ischemia was associated with histological evidence of caspase-3 activation, reactive astrogliosis, microglial activation, and motor neuron loss starting around 24–48h after spinal cord ischemia. Caspase-3 deficiency prevented delayed paraplegia and motor neuron loss after 5 min of spinal cord ischemia, but not immediate paraplegia after 9 min of ischemia.
Conclusion
The present results suggest that caspase-3 activation is required for delayed paraplegia and motor neuron degeneration after spinal cord ischemia.
doi:10.1161/STROKEAHA.110.600429
PMCID: PMC3199137  PMID: 21700940
Spinal cord ischemia; Delayed paraplegia; Delayed neuronal death; Cleaved caspase-3; Apoptosis
16.  Optical coherence tomography for the quantitative study of cerebrovascular physiology 
Doppler optical coherence tomography (DOCT) and OCT angiography are novel methods to investigate cerebrovascular physiology. In the rodent cortex, DOCT flow displays features characteristic of cerebral blood flow, including conservation along nonbranching vascular segments and at branch points. Moreover, DOCT flow values correlate with hydrogen clearance flow values when both are measured simultaneously. These data validate DOCT as a noninvasive quantitative method to measure tissue perfusion over a physiologic range.
doi:10.1038/jcbfm.2011.19
PMCID: PMC3130321  PMID: 21364599
hydrogen clearance; optical coherence tomography; optical imaging; vascular biology
17.  eNOS Protects from Atherosclerosis Despite Relevant Superoxide Production by the Enzyme in apoE−/− Mice 
PLoS ONE  2012;7(1):e30193.
Background
All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme.
Principal Findings
Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE−/−/eNOS−/−), while P/E-interactions did not differ, compared to apoE−/−. eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE−/− vessels.
Conclusion
Overt plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE−/−/eNOS−/− vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE−/− atherosclerosis but does not negate the enzyme's strong protective effects.
doi:10.1371/journal.pone.0030193
PMCID: PMC3264598  PMID: 22291917
18.  Endothelial nitric oxide synthase transgenic models of endothelial dysfunction 
Endothelial production of nitric oxide is critical to the regulation of vascular responses, including vascular tone and regional blood flow, leukocyte–endothelial interactions, platelet adhesion and aggregation, and vascular smooth muscle cell proliferation. A relative deficiency in the amount of bioavailable vascular NO results in endothelial dysfunction, with conditions that are conducive to the development of atherosclerosis: thrombosis, inflammation, neointimal proliferation, and vasoconstriction. This review focuses on mouse models of endothelial dysfunction caused by direct genetic modification of the endothelial nitric oxide synthase (eNOS) gene. We first describe the cardiovascular phenotypes of eNOS knockout mice, which are a model of total eNOS gene deficiency and thus the ultimate model of endothelial dysfunction. We then describe S1177A and S1177D eNOS mutant mice as mouse models with altered eNOS phosphorylation and therefore varying degrees of endothelial dysfunction. These include transgenic mice that carry the eNOS S1177A and S1177D transgenes, as well as knockin mice in which the endogenous eNOS gene has been mutated to carry the S1177A and S1177D mutations. Together, eNOS knockout mice and eNOS S1177 mutant mice are useful tools to study the effects of total genetic deficiency of eNOS as well as varying degrees of endothelial dysfunction caused by eNOS S1177 phosphorylation.
doi:10.1007/s00424-010-0867-4
PMCID: PMC2975487  PMID: 20697735
Nitric oxide; Endothelium; Vascular dysfunction; Transgenic mouse; Cerebral circulation
19.  Soluble Guanylate Cyclase α1β1 Limits Stroke Size and Attenuates Neurological Injury 
Background and Purpose
Nitric oxide (NO) mediates endothelium-dependent vasodilation, modulates cerebral blood flow (CBF), and determines stroke outcome. NO signals in part by stimulating soluble guanylate cyclase (sGC) to synthesize cGMP. To study the role of sGC in stroke injury, we compared the outcome of cerebral ischemia and reperfusion in mice deficient in the α1 subunit of sGC (sGCα1−/−) to that on wild-type (WT) mice.
Methods
Blood pressure, cerebrovascular anatomy, and vasoreactivity of pressurized carotid arteries were compared in both mouse genotypes. CBF was measured before and during middle cerebral artery (MCA) occlusion and reperfusion. We then assessed neurological deficit and infarct volume after 1 hour of occlusion and 23 hours of reperfusion, and after 24 hours of occlusion.
Results
Blood pressure and cerebrovascular anatomy were similar between genotypes. We found that vasodilation of carotid arteries in response to acetylcholine or sodium nitroprusside was diminished in sGCα1−/− compared to WT mice. CBF deficits did not differ between the genotypes during occlusion, but during reperfusion, CBF was 45% less in sGCα1−/− mice. Infarct volumes and neurological deficits were similar after 24 hours of occlusion in both genotypes. After 1 hour of ischemia and 23 hours of reperfusion, infarct volumes were two-fold larger and neurological deficits were worse in sGCα1−/− than in the WT mice.
Conclusion
sGCα1 deficiency impairs vascular reactivity to NO, and is associated with incomplete reperfusion, larger infarct size and worse neurological damage, suggesting that cGMP generated by sGCα1β1 is protective in ischemic stroke.
doi:10.1161/STROKEAHA.109.577635
PMCID: PMC3047459  PMID: 20595671
cerebral ischemia; gene knockout mice; mouse models
20.  Loss of insulin signaling in vascular endothelial cells accelerates atherosclerosis in apolipoprotein E null mice 
Cell metabolism  2010;11(5):379-389.
Summary
To determine whether insulin action on endothelial cells promotes or protects against atherosclerosis, we generated apolipoprotein E null mice in which the insulin receptor gene was intact or conditionally deleted in vascular endothelial cells. Insulin sensitivity, glucose tolerance, plasma lipids, and blood pressure were not different between the two groups, but atherosclerotic lesion size was more than 2-fold higher in mice lacking endothelial insulin signaling. Endothelium-dependent vasodilation was impaired and endothelial cell VCAM-1 expression was increased in these animals. Adhesion of mononuclear cells to endothelium in vivo was increased 4-fold compared with controls, but reduced to below control values by a VCAM-1 blocking antibody. These results provide definitive evidence that loss of insulin signaling in endothelium, in the absence of competing systemic risk factors, accelerates atherosclerosis. Therefore, improving insulin sensitivity in the endothelium of patients with insulin resistance or type 2 diabetes may prevent cardiovascular complications.
doi:10.1016/j.cmet.2010.03.013
PMCID: PMC3020149  PMID: 20444418
21.  Expression of neuronal nitric oxide synthase splice variants in atherosclerotic plaques of apoE knockout mice 
Atherosclerosis  2009;206(2):383-389.
Objective
We previously reported that deletion of brain type neuronal nitric oxide synthase-α (nNOS-α) accelerates atherosclerosis in apolipoproteinE (apoE) knockout (ko) mice. The regulation of nNOS expression is complex, involving the generation of mRNA splice variants. The current study investigates occurrence and distribution of nNOS variants in atherosclerotic lesions of apoE ko and apoE/nNOS-α double ko (dko) animals.
Methods
Mice were fed a high fat diet for 20 weeks. Immunohistochemistry and Western blot analysis were performed using antibodies detecting the carboxy terminal-, or amino terminal-residue of the nNOS protein. Confocal microscopy and in situ hybridization were used to identify the compartment of cellular expression.
Results
In situ hybridization revealed the presence of nNOS-α and -γ mRNA variants in apoE ko plaques, while only nNOS-γ was detectable in apoE/nNOS dko plaques. Consistent with mRNA expression nNOS-α protein can be detected in the neointima of apoE ko, but not apoE/nNOS dko animals. In contrast, the carboxy terminal antibody stained the neointima and media in apoE ko vessels and showed residual nNOS immunoreactivity in apoE/nNOS dko lesions. Confocal microscopy showed predominant nNOS expression in vascular smooth muscle cells, while colocalization with macrophages was less pronounced.
Conclusions
Our study shows that nNOS-α and -γ splice variants are expressed in atherosclerotic plaques of apoE ko mice. nNOS variants colocalized with markers for vascular smooth muscle cells and macrophages but not for endothelial cells. Since nNOS-α is atheroprotective, other nNOS splice variants which differ in enzyme kinetic and subcellular localization may also influence plaque formation.
doi:10.1016/j.atherosclerosis.2009.02.033
PMCID: PMC2757486  PMID: 19358992
Atherosclerosis; Neuronal nitric oxide synthase; Splice variants; Nitric oxide; nNOS variants
22.  eNOS, metabolic syndrome and cardiovascular disease 
Large epidemiologic studies have established that diabetes, hyperlipidemia and obesity all increase the risk for cardiovascular disease. However, the precise mechanisms by which these metabolic disorders increase the propensity to develop atherosclerosis are not known. Recently, the concept of the metabolic syndrome – a constellation of conditions including obesity, hypertension, hyperlipidemia and insulin resistance – has received much attention. Studies on the metabolic syndrome might enable a better understanding of the underlying biological mechanisms that lead to cardiovascular disease. This review focuses on endothelial nitric oxide synthase and summarizes evidence that a reduction in the bioavailability of endothelium-derived nitric oxide serves as a key link between metabolic disorders and cardiovascular risk.
doi:10.1016/j.tem.2009.03.005
PMCID: PMC2731551  PMID: 19647446
23.  Protective Effects of Statins after Embolic Focal Cerebral Ischemia in Endothelial Nitric Oxide Synthase Knockout Mice 
Previous studies have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) protect brain against ischemic injury by upregulating endothelial nitric oxide synthase (eNOS). Here, we tested the hypothesis that statins provide additional beneficial effects by upregulating endogenous tissue plasminogen activator (tPA) and by enhancing clot lysis in a model of embolic focal ischemia. Heterologous blood clots (0.2 mm) were injected into the distal internal carotid artery to occlude blood flow in the middle cerebral artery territory after chronic (14 days) simvastatin, atorvastatin or vehicle treatment . Ischemic lesion volume, neurologic deficits, as well as residual blood clots were measured at 22 hrs. RT-PCR assessed mRNA levels of eNOS, tPA, and the endogenous plasminogen activator inhibitor PAI-1. To show that statins protect via eNOS-independent mechanisms, experiments were also performed in eNOS null mice. Ischemic lesion volumes and neurologic deficits were significantly reduced in wild type mice by both simvastatin and atorvastatin. The protective effect was present regardless of whether clots were prepared from untreated or statin-treated mice, although the integrity and extent of the residual blood clot was less when statins were given as pretreatment. Statins increased eNOS and tPA mRNA levels but did not change mRNA levels of PAI-1. In eNOS knockout mice, atorvastatin reduced the volume of ischemic tissue and improved neurologic outcomes following vascular occlusion by blood clot emboli. These results suggest that statins protect against embolic stroke by mechanisms that appear eNOS independent. The beneficial actions may relate in part, to enhanced clot lysis due to upregulation of endogenous tPA levels and/or decreased thrombosis.
doi:10.1038/sj.jcbfm.9600070
PMCID: PMC2742229  PMID: 15716855
24.  Cardiovascular roles of nitric oxide: A review of insights from nitric oxide synthase gene disrupted mice† 
Cardiovascular research  2008;77(1):19-29.
Nitric oxide (NO) is a gaseous molecule that plays many key roles in the cardiovascular system. Each of the enzymes that generate NO—neuronal, inducible and endothelial NO synthase—has been genetically disrupted in mice. This review discusses the cardiovascular phenotypes of each of the NO synthase (NOS) gene knockout mice, and the insights gained into the roles of NO in the cardiovascular system. Mice lacking the endothelial isoform are hypertensive, have endothelial dysfunction and show a more severe outcome in response to vascular injury, to stroke and cerebral ischaemia, and to diet-induced atherosclerosis. Mice lacking the neuronal isoform show a less severe outcome in response to stroke and cerebral ischaemia but have increased diet-induced atherosclerosis. Mice lacking the inducible isoform show reduced hypotension to septic shock. Together, NOS gene knockout mice have been useful tools that complement our other approaches to studying the multiple roles of NO in the cardiovascular system.
doi:10.1016/j.cardiores.2007.06.024
PMCID: PMC2731989  PMID: 17658499
Nitric oxide; Gene knockout; Targeted disruption; Animal models; Pathophysiology; Atherosclerosis; Vascular dysfunction; Cerebral ischaemia; Mouse models
25.  Contributions of nitric oxide synthase isoforms to pulmonary oxygen toxicity, local vs. mediated effects 
Reactive species of oxygen and nitrogen have been collectively implicated in pulmonary oxygen toxicity, but the contributions of specific molecules are unknown. Therefore, we assessed the roles of several reactive species, particularly nitric oxide, in pulmonary injury by exposing wild-type mice and seven groups of genetically altered mice to >98% O2 at 1, 3, or 4 atmospheres absolute. Genetically altered animals included knockouts lacking either neuronal nitric oxide synthase (nNOS−/−), endothelial nitric oxide synthase (eNOS−/−), inducible nitric oxide synthase (iNOS−/−), extracellular superoxide dismutase (SOD3−/−), or glutathione peroxidase 1 (GPx1−/−), as well as two transgenic variants (S1179A and S1179D) having altered eNOS activities. We confirmed our earlier finding that normobaric hyperoxia (NBO2) and hyperbaric hyperoxia (HBO2) result in at least two distinct but overlapping patterns of pulmonary injury. Our new findings are that the role of nitric oxide in the pulmonary pathophysiology of hyperoxia depends both on the specific NOS isozyme that is its source and on the level of hyperoxia. Thus, iNOS predominates in the etiology of lung injury in NBO2, and SOD3 provides an important defense. But in HBO2, nNOS is a major contributor to pulmonary injury, whereas eNOS is protective. In addition, we demonstrated that nitric oxide derived from nNOS is involved in a neurogenic mechanism of HBO2-induced lung injury that is linked to central nervous system oxygen toxicity through adrenergic/cholinergic pathways.
doi:10.1152/ajplung.00420.2007
PMCID: PMC2728469  PMID: 18326824
normobaric oxygen toxicity; hyperbaric oxygen toxicity; superoxide dismutase; glutathione peroxidase 1; neurogenic pulmonary oxygen toxicity

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