Article

The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo

Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Boston, MA 02129, USA.
Journal of Clinical Investigation (Impact Factor: 13.77). 08/2007; 117(7):1961-7. DOI: 10.1172/JCI29877
Source: PubMed

ABSTRACT 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.

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Available from: Salvatore Salomone, Aug 28, 2015
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    • "Endothelial nitric oxide synthase (eNOS) is an important enzyme that exists in many tissues and organs. It serves to generate nitric oxide (NO), which regulates vascular tone, angiogenesis, and multiple inflammatory signaling pathways [1], [2], [3]. Two identical eNOS monomers can associate to form a dimer. "
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    ABSTRACT: Endothelial nitric oxide synthase (eNOS) is a multifunctional enzyme with roles in diverse cellular processes including angiogenesis, tissue remodeling, and the maintenance of vascular tone. Monomeric and dimeric forms of eNOS exist in various tissues. The dimeric form of eNOS is considered the active form and the monomeric form is considered inactive. The activity of eNOS is also regulated by many other mechanisms, including amino acid phosphorylation and interactions with other proteins. However, the precise mechanisms regulating eNOS dimerization, phosphorylation, and activity remain incompletely characterized. We utilized purified eNOS and bovine aorta endothelial cells (BAECs) to investigate the mechanisms regulating eNOS degradation. Both eNOS monomer and dimer existed in purified bovine eNOS. Incubation of purified bovine eNOS with protein phosphatase 2A (PP2A) resulted in dephosphorylation at Serine 1179 (Ser1179) in both dimer and monomer and decrease in eNOS activity. However, the eNOS dimer∶monomer ratio was unchanged. Similarly, protein phosphatase 1 (PP1) induced dephosphorylation of eNOS at Threonine 497 (Thr497), without altering the eNOS dimer∶monomer ratio. Different from purified eNOS, in cultured BAECs eNOS existed predominantly as dimers. However, eNOS monomers accumulated following treatment with the proteasome inhibitor lactacystin. Additionally, treatment of BAECs with vascular endothelial growth factor (VEGF) resulted in phosphorylation of Ser1179 in eNOS dimers without altering the phosphorylation status of Thr497 in either form. Inhibition of heat shock protein 90 (Hsp90) or Hsp90 silencing destabilized eNOS dimers and was accompanied by dephosphorylation both of Ser1179 and Thr497. In conclusion, our study demonstrates that eNOS monomers, but not eNOS dimers, are degraded by ubiquitination. Additionally, the dimeric eNOS structure is the predominant condition for eNOS amino acid modification and activity regulation. Finally, destabilization of eNOS dimers not only results in eNOS degradation, but also causes changes in eNOS amino acid modifications that further affect eNOS activity.
    PLoS ONE 08/2014; 9(8):e105479. DOI:10.1371/journal.pone.0105479 · 3.23 Impact Factor
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    • "Here, we use eNOS mutant mice that carry the S1176D gain of function mutation [23]. We previously showed that this phosphomimetic mutation rescues impaired blood flow in Akt1 deficient mice subjected to wound healing assays [37], and improves vessel reactivity and decreases stroke size when challenged with cerebral ischemia [38]. "
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    ABSTRACT: 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. 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. 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.
    PLoS ONE 01/2014; 9(1):e85946. DOI:10.1371/journal.pone.0085946 · 3.23 Impact Factor
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    • "Silicon-covered nylon filament (Doccol) was introduced into the external carotid artery with MCAO with subsequent reperfusion and advanced to the middle cerebral artery for 30 min. as described elsewhere [21], [22]. All procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Massachusetts General Hospital Subcommittee on Research and Animal Care (Permit Number: 2003N000297). "
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    ABSTRACT: 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.
    PLoS ONE 10/2013; 8(10):e77002. DOI:10.1371/journal.pone.0077002 · 3.23 Impact Factor
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