David Fulton

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals

Recent studies suggest that statins can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. We show here that statins rapidly activate the protein kinase Akt/PKB in endothelial cells. Accordingly, simvastatin enhanced phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS), inhibited apoptosis and accelerated vascular structure formation in vitro in an Akt-dependent manner. Similar to vascular endothelial growth factor (VEGF) treatment, both simvastatin administration and enhanced Akt signaling in the endothelium promoted angiogenesis in ischemic limbs of normocholesterolemic rabbits. Therefore, activation of Akt represents a mechanism that can account for some of the beneficial side effects of statins, including the promotion of new blood vessel growth.

The Treatment of Kawasaki Syndrome with Intravenous Gamma Globulin

We compared the efficacy of intravenous gamma globulin plus aspirin with that of aspirin alone in reducing the frequency of coronary-artery abnormalities in children with acute Kawasaki syndrome in a multicenter, randomized trial. Children randomly assigned to the gamma globulin group received intravenous gamma globulin, 400 mg per kilogram of body weight per day, for four consecutive days; both treatment groups received aspirin, 100 mg per kilogram per day, through the 14th day of illness, then 3 to 5 mg per kilogram per day. Two-dimensional echocardiograms were interpreted blindly and independently by two or more readers. Two weeks after enrollment, coronary-artery abnormalities were present in 18 of 78 children (23 percent) in the aspirin group, as compared with 6 of 75 (8 percent) in the gamma globulin group (P = 0.01). Seven weeks after enrollment, abnormalities were present in 14 of 79 children (18 percent) in the aspirin group and in 3 of 79 (4 percent) in the gamma globulin group (P = 0.005). No child had serious adverse effects from receiving gamma globulin. We conclude that high-dose intravenous gamma globulin is safe and effective in reducing the prevalence of coronary-artery abnormalities when administered early in the course of Kawasaki syndrome.

A Single Intravenous Infusion of Gamma Globulin as Compared with Four Infusions in the Treatment of Acute Kawasaki Syndrome

BACKGROUND Treatment of acute Kawasaki syndrome with a four-day course of intravenous gamma globulin, together with aspirin, has been demonstrated to be safe and effective in preventing coronary-artery lesions and reducing systemic inflammation. We hypothesized that therapy with a single, very high dose of gamma globulin would be at least as effective as the standard regimen. METHODS We conducted a multicenter, randomized, controlled trial involving 549 children with acute Kawasaki syndrome. The children were assigned to receive gamma globulin either as a single infusion of 2 g per kilogram of body weight over 10 hours or as daily infusions of 400 mg per kilogram for four consecutive days. Both treatment groups received aspirin (100 mg per kilogram per day through the 14th day of illness, then 3 to 5 mg per kilogram per day). RESULTS The relative prevalence of coronary abnormalities, adjusted for age and sex, among patients treated with the four-day regimen, as compared with those treated with the single-infusion regimen, was 1.94 (95 percent confidence limits, 1.01 and 3.71) two weeks after enrollment and 1.84 (95 percent confidence limits, 0.89 and 3.82) seven weeks after enrollment. Children treated with the single-infusion regimen had lower mean temperatures while hospitalized (day 2, P less than 0.001; day 3, P = 0.004), as well as a shorter mean duration of fever (P = 0.028). Furthermore, in the single-infusion group the laboratory indexes of acute inflammation moved more rapidly toward normal, including the adjusted serum albumin level (P = 0.004), alpha 1-antitrypsin level (P = 0.007), and C-reactive protein level (P = 0.017). Lower IgG levels on day 4 were associated with a higher prevalence of coronary lesions (P = 0.005) and with a greater degree of systemic inflammation. The two groups had a similar incidence of adverse effects (including new or worsening congestive heart failure in nine children), which occurred in 2.7 percent of the children overall. All the adverse effects were transient. CONCLUSIONS In children with acute Kawasaki disease, a single large dose of intravenous gamma globulin is more effective than the conventional regimen of four smaller daily doses and is equally safe.

Membrane Estrogen Receptor Engagement Activates Endothelial Nitric Oxide Synthase via the PI3-Kinase–Akt Pathway in Human Endothelial Cells

17&bgr;-Estradiol (E2) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E2-stimulated endothelial NO release can occur without an increase in cytosolic Ca2+. Here we demonstrate for the first time, to our knowledge, that E2 rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase–Akt pathway. E2 treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182,780. E2 stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E2-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca2+ levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E2-stimulated NO release. The membrane-impermeant E2BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase–Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca2+ fluxes and describes an important pathway relevant to cardiovascular pathophysiology.

Vascular Endothelial Growth Factor–Stimulated Actin Reorganization and Migration of Endothelial Cells Is Regulated via the Serine/Threonine Kinase Akt

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.

Domain Mapping Studies Reveal That the M Domain of hsp90 Serves as a Molecular Scaffold to Regulate Akt-Dependent Phosphorylation of Endothelial Nitric Oxide Synthase and NO Release

Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation.

Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production.

There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were “uncoupling” NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.

A new role for Nogo as a regulator of vascular remodeling

Although Nogo-A has been identified in the central nervous system as an inhibitor of axonal regeneration, the peripheral roles of Nogo isoforms remain virtually unknown. Here, using a proteomic analysis to identify proteins enriched in caveolae and/or lipid rafts (CEM/LR), we show that Nogo-B is highly expressed in cultured endothelial and smooth muscle cells, as well as in intact blood vessels. The N terminus of Nogo-B promotes the migration of endothelial cells but inhibits the migration of vascular smooth muscle (VSM) cells, processes necessary for vascular remodeling. Vascular injury in Nogo-A/B-deficient mice promotes exaggerated neointimal proliferation, and adenoviral-mediated gene transfer of Nogo-B rescues the abnormal vascular expansion in those knockout mice. Our discovery that Nogo-B is a regulator of vascular homeostasis and remodeling broadens the functional scope of this family of proteins.

Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking

Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-nitrosylation. Although intracellular S-nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein S-nitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimide-sensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-nitrosylation reactions that can regulate intracellular transport processes.