TNFα reduces eNOS activity in endothelial cells through serine 116 phosphorylation and Pin1 binding: Confirmation of a direct, inhibitory interaction of Pin1 with eNOS.

Production of NO by the endothelial nitric oxide synthase (eNOS) has a major role in blood pressure control and suppression of atherosclerosis. In a previous study, we presented evidence implicating the Pin1 prolyl isomerase in negative modulation of eNOS activity in bovine aortic endothelial cells (BAECs). Pin1 recognizes phosphoserine/phosphothreonine-proline motifs in target proteins and catalyzes prolyl isomerization at the peptide bond. In the present study, we show, first, with purified proteins, that Pin1 binds to eNOS directly via the Pin1 WW domain. Binding is enhanced by mimicking phosphorylation of eNOS at S116. Interaction of Pin1 with eNOS markedly reduces eNOS enzymatic activity. Second, in BAECs, we show that TNFα induces ERK 1/2-mediated S116 phosphorylation of eNOS, accompanied by Pin1 binding. TNFα treatment of BAECs results in a reduction in NO release from the cells in a manner that depends on the activities of both Pin1 and ERK 1/2. Evidence is also presented that this mechanism of eNOS regulation cannot occur in rat and mouse cells because there is no proline residue in the mouse and rat amino acid sequences adjacent to the putative phosphorylation site. Moreover, we find that phosphorylation of this site is not detectable in mouse eNOS.

Phytonutrients Differentially Stimulate NAD(P)H:Quinone Oxidoreductase, Inhibit Proliferation, and Trigger Mitotic Catastrophe in Hepa1c1c7 Cells.

Phytonutrients have rapidly emerged as natural food chemicals possessing multifaceted biological actions that may support beneficial health outcomes. Among the vast array of phytonutrients currently being studied, sulforaphane, curcumin, quercetin, and resveratrol have been frequently reported to stimulate the expression of endogenous detoxification enzymes and may thereby facilitate the neutralization of otherwise harmful environmental agents. Some of these same phytonutrients, however, have also been implicated in disrupting normal cell proliferation and hence may possess toxic properties in and of themselves. In this study, we characterize the respective minimum threshold concentrations of the aforementioned phytonutrients in Hepa1c1c7 cells that stimulate NAD(P)H: quinone oxidoreductase (NQO1), a key enzyme in the hepatic neutralization of menadione, other biological oxidants, and some environmental carcinogens. Moreover, our findings demonstrate that relatively low concentrations of either sulforaphane or curcumin significantly (P < .05) increase NQO1 protein expression and activity without triggering G2/M cell cycle arrest or mitotic catastrophe. The minimal quercetin concentration inducing NQO1, however, was 100-fold higher than that which disrupted mitosis. Also, while resveratrol modestly stimulated NQO1, the minimally effective resveratrol concentration concomitantly induced evidence of cellular apoptosis. Taken together, these findings indicate that only particular phytonutrients are likely efficacious in upregulating NQO1 activity without also leading to hepatic cytotoxicity.

Amyloid ß peptide-induced inhibition of endothelial nitric oxide production involves oxidative stress-mediated constitutive eNOS/HSP90 interaction and disruption of agonist-mediated Akt activation.

BACKGROUND: Amyloid ß (Aß)-induced vascular dysfunction significantly contributes to the pathogenesis of Alzheimer's disease (AD). Aß is known to impair endothelial nitric oxide synthase (eNOS) activity, thus inhibiting endothelial nitric oxide production (NO). METHOD: In this study, we investigated Aß-effects on heat shock protein 90 (HSP90) interaction with eNOS and Akt in cultured vascular endothelial cells and also explored the role of oxidative stress in this process. RESULTS: Treatments of endothelial cells (EC) with Aß promoted the constitutive association of HSP90 with eNOS but abrogated agonist (vascular endothelial growth factor (VEGF))-mediated HSP90 interaction with Akt. This effect resulted in blockade of agonist-mediated phosphorylation of Akt and eNOS at serine 1179. Furthermore, Aß stimulated the production of reactive oxygen species in endothelial cells and concomitant treatments of the cells with the antioxidant N-acetyl-cysteine (NAC) prevented Aß effects in promoting HSP90/eNOS interaction and rescued agonist-mediated Akt and eNOS phosphorylation. CONCLUSIONS: The obtained data support the hypothesis that oxidative damage caused by Aß results in altered interaction of HSP90 with Akt and eNOS, therefore promoting vascular dysfunction. This mechanism, by contributing to Aß-mediated blockade of nitric oxide production, may significantly contribute to the cognitive impairment seen in AD patients.

Curcumin binds tubulin, induces mitotic catastrophe, and impedes normal endothelial cell proliferation.

Curcumin, a component of turmeric spice that imparts flavor and color to curry, is thought to possess anti-inflammatory and antioxidant properties in biological tissues. However, while such efficacies have been described in the context of carcinogenesis, the impact of curcumin on normal cell cycle regulation is poorly understood. Here, we provide evidence of curcumin toxicity in proliferating bovine aortic endothelial cells, at concentrations relevant to the diet and below those previously reported in cancer models. Upon confirming curcumin's ability to upregulate hemeoxygenase-1 in a dose-dependent fashion, we found the minimally efficacious curcumin concentration to also inhibit endothelial cell DNA synthesis. Moreover, curcumin concentrations below the minimum 2 µM threshold required to induce hemeoxygenase-1 bound tubulin protein in vitro and triggered hallmark evidence of mitotic catastrophe in vivo. Concentrations as low as 0.1 µM curcumin led to disproportionate DNA segregation, karyorrhexis, and micronucleation in proliferating endothelial cells. While suggesting a mechanism by which physiological curcumin concentrations inhibit cell cycle progression, these findings describe heretofore unappreciated curcumin toxicity with potential implications for endothelial growth, development, and tissue healing.

Calcineurin-mediated dephosphorylation of eNOS at serine 116 affects eNOS enzymatic activity indirectly by facilitating c-Src binding and tyrosine 83 phosphorylation.

It has been shown previously that phosphorylation of the endothelial nitric oxide synthase (eNOS) at serine 116 (S116) under basal conditions suppresses eNOS enzymatic activity in endothelial cells. It has also been shown that vascular endothelial growth factor (VEGF) treatment of endothelial cells produces a rapid S116 dephosphorylation, which is blocked by the calcineurin inhibitor, cyclosporin A (CsA). In this study, we show that activation of eNOS in response to a variety of other eNOS-activating agonists and the cytosolic calcium-elevating agent, thapsigargin also involves CsA-inhibitable S116 dephosphorylation. Studies with the purified eNOS enzyme also demonstrate that neither mimicking phosphorylation at S116 nor phosphorylation of the purified enzyme at S116 in vitro has any effect on enzymatic activity. Phospho-mimicking, however, does interfere with the interaction of eNOS with c-Src, an interaction which is known to activate eNOS by phosphorylation at tyrosine 83 (Y83). Agonist-stimulated eNOS-Src complex formation, as well as agonist-stimulated Y83 phosphorylation, are blocked by calcineurin inhibition by CsA and by a cell-permeable calcineurin inhibitory peptide. Taken together, these data suggest a mechanism of eNOS regulation whereby calcineurin-mediated dephosphorylation of eNOS at S116 affects eNOS enzymatic activity indirectly, rather than directly, by facilitating c-Src binding and Y83 phosphorylation.

Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation.

The NADPH oxidases (Noxs) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of this study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and this inhibition was reversible with chronic, but not acute, exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, and eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation, or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO sensitivity, and biotin labeling. Furthermore, coexpression of the denitrosylation enzymes thioredoxin 1 and GSNO reductase prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was in the order Nox1 ≥ Nox3 > Nox5 > Nox2, whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5.

Expression and functional significance of NADPH oxidase 5 (Nox5) and its splice variants in human blood vessels.

The expression and functional significance of NADPH oxidase 5 (Nox5) and its five isoforms in vascular cells is poorly understood. The goal of this study was to determine whether Nox5-α, -ß, -δ, -γ, and -ε (short) are expressed in human blood vessels and evaluate their respective functions. Nox5 mRNA and protein were detected in human blood vessels, cultured human vascular smooth muscle (HVSMC) and endothelium, but not fibroblasts. The most abundant isoforms were α and ß, whereas δ and γ were not detected. Nox5-α and -ß produced reactive oxygen species (ROS), but -δ, -γ, and -ε were not catalytically active. Coexpression of the active Nox5 isoforms with inactive Nox5 variants suppressed ROS production, and coimmunoprecipitation revealed that Nox5-ß binds the inactive ε variant, which may account for reduced ROS production. In HVSMC, angiotensin II, endothelin-1 and TNF-α increased endogenous Nox5 mRNA levels, while adenovirus-mediated overexpression of Nox5 promoted p38 MAPK, JAK2, JNK, and ERK1/2 phosphorylation in endothelial cells (EC), but only increased ERK1/2 phosphorylation in HVSMC. At higher levels of Nox5, there was evidence of increased apoptosis in EC, but not in HVSMC, as detected by the presence of cleaved caspase-3 and cleaved poly(ADP-ribose)polymerase. Although catalytically inactive, Nox5-ε potently activated ERK in HVSMC, and increased expression of Nox5-ε promoted HVSMC proliferation. Nox5 is expressed in human blood vessels. The Nox5-α and -ß splice variants are the major isoforms that are expressed and the only variants capable of ROS production. Nox5-ε can inhibit Nox5 activity and activate ERK and HVSMC proliferation.

Pin1 prolyl isomerase regulates endothelial nitric oxide synthase.

OBJECTIVE: The Pin1 prolyl isomerase acts in concert with proline-directed protein kinases to regulate function of protein substrates through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline. We sought to determine whether Pin1 interacts with endothelial nitric oxide synthase (eNOS) in endothelial cells in a manner that depends on proline-directed phosphorylation of the eNOS enzyme and whether this interaction influences basal or agonist-stimulated eNOS activity. METHODS AND RESULTS: Inhibitors of the extracellular-regulated kinase (ERK) 1/2 MAP kinases inhibit proline-directed phosphorylation of eNOS at serine 116 (Ser116) in bovine aortic endothelial cells (BAECs). Moreover, eNOS and Pin1 can be coimmunoprecipitated from BAECs only when Ser116 is phosphorylated. In addition, phosphomimetic Ser116Asp eNOS, but not wild-type eNOS, can be coimmunoprecipitated with Pin1 coexpressed in COS-7 cells. Inhibition of Pin1 in BAECs by juglone or by dominant negative Pin1 increases basal and agonist-stimulated NO release from the cells, whereas overexpression of wild-type Pin1 in BAECs suppresses basal and agonist-stimulated NO production. Overexpression of wild-type Pin1 in intact aortae also reduces agonist-induced relaxation of aortic rings. CONCLUSIONS: Our results demonstrate a novel form of eNOS regulation in endothelial cells and blood vessels through Ser116 phosphorylation-dependent interaction of eNOS with Pin1.

Activation of endothelial nitric oxide synthase by the pro-apoptotic drug embelin: Striking discrepancy between nitric oxide-mediated cyclic GMP accumulation and L-citrulline formation.

The benzoquinone derivative embelin is a multifunctional drug that not only induces apoptosis by inhibiting XIAP, the X chromosome-linked inhibitor of apoptosis protein, but also blocks nuclear factor-kappaB signaling pathways, thereby leading to down-regulation of a variety of gene products involved in tumor cell survival, proliferation, invasion, angiogenesis, and inflammation. Here, we report that embelin activates and modulates l-arginine/nitric oxide/cyclic GMP signaling in cultured endothelial cells. Embelin elicited a rapid increase of intracellular free Ca(2+), leading to activation of endothelial nitric oxide synthase (eNOS) and NO-induced cGMP accumulation. While the cGMP response was comparable to that caused by other Ca(2+)-mobilizing agents, the stimulatory effect of embelin on l-citrulline formation (approximately 4-fold) was substantially lower than that observed upon activation of eNOS with the Ca(2+) ionophore A23187 (approximately 18-fold), the receptor agonist ATP (approximately 16-fold) or the sarco-endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (approximately 14-fold). The apparent discrepancy between NO/cGMP and l-citrulline formation in embelin-treated cells was not due to enhanced metabolism and/or efflux of l-citrulline, increased NO bioavailability, inhibition of cGMP hydrolysis, sensitization of soluble guanylate cyclase (sGC) to NO, or enhanced formation of a sGC/eNOS complex. Our puzzling observations suggest that embelin improves coupling of endothelial NO synthesis to sGC activation through mobilization of an as yet unrecognized signaling pathway.

Inhibition of endothelial nitric oxide synthase by the lipid phosphatase PTEN.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a lipid phosphatase that functions as a negative regulator of the phosphoinositide-3-kinase (PI3K) pathway. The present study sought to examine in depth the interaction between PTEN and eNOS activity. Co-expression of eNOS and PTEN in COS-7 cells significantly decreased NO production compared to eNOS alone, while co-expression of eNOS and the dominant negative mutant PTEN(C124A) significantly increased NO production. Upon examination of the putative eNOS phosphorylation sites, phosphorylation of S116, T497, S617, S635 and S1179 was decreased by PTEN co-expression, while the dominant negative PTEN(C124A) produced an increase in phosphorylation of all sites except S116 and S635. A myristoylation-deficient eNOS construct with little dependence on phosphorylation state (G2AeNOS) was not significantly affected by co-expression with either PTEN or PTEN(C124A). Likewise, an eNOS construct with a triple phospho-null mutation (S617A, S635A and S1179A) was also unaffected by co-expression with either PTEN or PTEN(C124A). Purified PTEN or PTEN(C124A) failed to interact with purified eNOS in vitro, arguing against a direct interaction between PTEN and eNOS. When the PTEN constructs were expressed in human aortic endothelial cells (HAECs), PTEN significantly decreased NO production and PTEN(C124A) increased it, and both S617 and S1179 were altered by co-expression with the PTEN constructs. Increased expression of PTEN in endothelial cells did not influence superoxide production. We conclude that PTEN is a regulator of eNOS function both when expressed in COS-7 cells and in human endothelial cells, and does so via its effects on the PI3K/Akt pathway.

Alterations in lung arginine metabolism in lambs with pulmonary hypertension associated with increased pulmonary blood flow.

Previous studies demonstrate impaired nitric oxide (NO) signaling in children and animal models with congenital heart defects and increased pulmonary blood flow. However, the molecular mechanisms underlying these alterations remain incompletely understood. The purpose of this study was to determine if early changes in arginine metabolic pathways could play a role in the reduced NO signaling demonstrated in our lamb model of congenital heart disease with increased pulmonary blood flow (Shunt lambs). The activities of the arginine recycling enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) were both decreased in lung tissues of Shunt lambs while arginase activity was increased. Associated with these alterations, lung L-arginine levels were decreased. These changes correlated with an increase in NO synthase-derived reactive oxygen species (ROS) generation. This study provides further insights into the molecular mechanisms leading to decreased NO signaling in Shunt lambs and suggests that altered arginine metabolism may play a role in the development of the endothelial dysfunction associated with pulmonary hypertension secondary to increased pulmonary blood flow.

The hnRNA-binding proteins hnRNP L and PTB are required for efficient translation of the Cat-1 arginine/lysine transporter mRNA during amino acid starvation.

The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5' untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat-1 IRES was important for PTB binding and for the induction of translation during amino acid starvation. Binding of hnRNP L to the IRES or the Cat-1 mRNA in vivo was independent of PTB binding but was not sufficient to increase IRES activity or Cat-1 mRNA translation during amino acid starvation. In contrast, binding of PTB to the Cat-1 mRNA in vivo required hnRNP L. A wider role of hnRNP L in mRNA translation was suggested by the decrease of global protein synthesis in cells with reduced hnRNP L levels. It is proposed that PTB and hnRNP L are positive regulators of Cat-1 mRNA translation via the IRES under stress conditions that cause a global decrease of protein synthesis.

Increased nitric oxide synthase activity and Hsp90 association in skeletal muscle following chronic exercise.

Exercise training results in dynamic changes in skeletal muscle blood flow and metabolism. Nitric oxide (NO) influences blood flow, oxidative stress, and glucose metabolism. Hsp90 interacts directly with nitric oxide synthases (NOS), increasing NOS activity and altering the balance of superoxide versus NO production. In addition, Hsp90 expression increases in various tissues following exercise. Therefore, we tested the hypothesis that exercise training increases Hsp90 expression as well as Hsp90/NOS association and NOS activity in skeletal muscle. Male, Sprague-Dawley rats were assigned to either a sedentary or exercise trained group (n = 10/group). Exercise training consisted of running on a motorized treadmill for 10 weeks at 30 m/min, 5% grade for 1 h. Western blotting revealed that exercise training resulted in a 1.9 +/- 0.1-fold increase in Hsp90 expression in the soleus muscle but no increase in neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase, or endothelial nitric oxide synthase (eNOS). Exercise training also resulted in a 3.4 +/- 1.0-fold increase in Hsp90 association with nNOS, a 2.3 +/- 0.4-fold increase association with eNOS measured by immunoprecipitation as well as a 1.5 +/- 0.3-fold increase in eNOS phosphorylation at Ser-1179. Total NOS activity measured by the rate of conversion of L-[(14)C]arginine to L-[(14)C]citrulline was increased by 1.42 +/- 0.9 fold in soleus muscle following exercise training compared to controls. In summary, a 10-week treadmill training program in rats results in a significant increase in total NOS activity in the soleus which may be due, in part, to increased NOS interaction with Hsp90 and phosphorylation. This interaction may play a role in altering muscle blood flow and skeletal muscle redox status.

An essential role for SRC-activated STAT-3 in 14,15-EET-induced VEGF expression and angiogenesis.

To understand the molecular mechanisms underlying 14,15-epoxyeicosatrienoic acid (14,15-EET)-induced angiogenesis, here we have studied the role of signal transducer and activator of transcription-3 (STAT-3). 14,15-EET stimulated the tyrosine phosphorylation of STAT-3 and its translocation from the cytoplasm to the nucleus in human dermal microvascular endothelial cells (HDMVECs). Adenovirus-mediated delivery of dominant negative STAT-3 substantially inhibited 14,15-EET-induced HDMVEC migration, and tube formation and Matrigel plug angiogenesis. 14,15-EET activated Src, as measured by its tyrosine phosphorylation and blockade of its activation by adenovirus-mediated expression of its dominant negative mutant, significantly attenuated 14,15-EET-induced STAT-3 phosphorylation in HDMVECs and the migration and tube formation of these cells and Matrigel plug angiogenesis. 14,15-EET induced the expression of vascular endothelial cell growth factor (VEGF) in a time- and Src-STAT-3-dependent manner in HDMVECs. Transfac analysis of VEGF promoter revealed the presence of STAT-binding elements and 14,15-EET induced STAT-3 binding to this promoter in vivo, and this interaction was inhibited by suppression of Src-STAT-3 signaling. Neutralizing anti-VEGF antibodies completely blocked 14,15-EET-induced HDMVEC migration and tube formation and Matrigel plug angiogenesis. These results reveal that Src-dependent STAT-3-mediated VEGF expression is a major mechanism of 14,15-EET-induced angiogenesis.

Agonist-stimulated endothelial nitric oxide synthase activation and vascular relaxation. Role of eNOS phosphorylation at Tyr83.

Tyr83 in endothelial nitric oxide synthase (eNOS) has been identified previously as a site of Src kinase-mediated phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) that is phosphorylated in response to oxidant stress. In the present study, we have used a phospho-specific antibody to show that Tyr83 in eNOS is also phosphorylated in both BAECs and intact blood vessel segments in response to treatment with a variety of different eNOS-activating agonists, including thapsigargin, vascular endothelial growth factor, bradykinin, ATP, sphingosine-1-phosphate, estrogen, angiopoietin, and acetylcholine. Agonist stimulation of eNOS Tyr83 phosphorylation as well as agonist stimulation of endothelial NO release in BAECs is blocked by Src kinase inhibition by either 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) or by dominant negative Src. Mutation of Tyr83 to a nonphosphorylatable Phe blocks agonist stimulation of NO release from eNOS-reconstituted eNOS knockdown endothelial cells. Mutation of Tyr83 also attenuates agonist-induced relaxation of eNOS-reconstituted aortic rings from eNOS knockout mice. Phosphorylation of eNOS at Tyr83 thus appears to be a common covalent modification that is induced, not only by oxidant stress but also by other physiologically relevant extracellular signals known to be important in regulation of eNOS activity in vivo. Moreover, our results demonstrate an important role for Src-mediated phosphorylation of eNOS at Tyr83 in agonist stimulation of eNOS activation and vascular relaxation.

Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase.

The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.

Role of eNOS phosphorylation at Ser-116 in regulation of eNOS activity in endothelial cells.

Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), an important modulator of vascular function. eNOS is regulated post-translationally through phosphorylation/dephosphorylation at a number of specific phosphorylation sites including Ser-116 in the bovine eNOS sequence. Whether phosphorylation of eNOS at Ser-116 in endothelial cells is stimulatory or inhibitory has not previously been definitively determined. In this study we show that mimicking phosphorylation of eNOS at Ser-116 by Asp mutation reduces basal NO release from endothelial cells. Preventing phosphorylation at this site by Ala mutation increases the amount of NO release from endothelial cells in response to agonist stimulation. In addition, mimicking phosphorylation of Ser-116 increases eNOS association with caveolin-1 and reduces the vascular reactivity of intact aortic rings. eNOS phosphorylation at Ser-116, therefore, appears to contribute to negative modulation of eNOS activity and hence to regulation of vascular tone.

Nitric oxide and superoxide generation from endothelial NOS: modulation by HSP90.

Previously, we have shown that pulmonary arterial endothelial cells (PAECs) isolated from fetal lambs produce significant levels of nitric oxide (NO) but minimal superoxide upon stimulation, whereas PAECs isolated from 4-wk-old lambs produce significant amounts of both NO and superoxide. These data indicated that a certain degree of uncoupling of endothelial NO synthase (eNOS) occurs in PAECs during postnatal development. In this study, we sought to extend these studies by investigating the potential role of heat shock protein 90 (HSP90) in eNOS coupling. Western blot analyses revealed higher HSP90 expression in PAECs isolated from fetal compared with 4-wk-old lambs, whereas the analysis of recombinant human eNOS activation in vitro in the presence of HSP90 indicated that HSP90 significantly augmented NO production while inhibiting superoxide generation from eNOS. To further investigate whether HSP90 could be involved in uncoupling of eNOS in PAECs isolated from 4-wk-old lambs, we utilized an adenovirus to overexpress HSP90. We found that overexpression of HSP90 significantly increased the shear-stimulated association of HSP90 with eNOS and led to significant increases in NO production and reduced NOS-dependent superoxide generation. Conversely, the exposure of PAECs isolated from fetal lambs to the HSP90 inhibitor radicicol led to significant decreases in eNOS-HSP90 interactions, decreased shear-stimulated NO generation, and increased NOS-dependent superoxide production indicative of eNOS uncoupling. Finally, we examined eNOS-HSP90 interactions in our lamb model of pulmonary hypertension associated with increased pulmonary blood flow (shunt). Our data indicate that HSP90-eNOS interactions were decreased in shunt lambs and that this was associated with decreased NO generation and an increase in eNOS-dependent generation of superoxide. Together, our data support a significant role for HSP90 in promoting NO generation and inhibiting superoxide generation by eNOS and indicate that the disruption of this interaction may be involved in the endothelial dysfunction associated with pulmonary hypertension.

Heat shock protein 90 inhibitors prolong survival, attenuate inflammation, and reduce lung injury in murine sepsis.

RATIONALE: Severe sepsis is the leading cause of death for patients in intensive care units. Patients with severe sepsis develop multiple organ failure, including acute lung injury (ALI), resulting from a deregulated inflammatory response. Inhibitors of the ubiquitous chaperone, heat shock protein 90 (Hsp90), block the activity of certain proinflammatory mediators in vitro. We hypothesized that Hsp90 inhibitors may ameliorate the inflammation and ALI associated with severe sepsis. OBJECTIVES: To test the hypothesis that Hsp90 inhibitors prolong survival, attenuate inflammation, and reduce lung injury in a murine model of sepsis. METHODS: Male C57BL/6 mice received either one of two Hsp90 inhibitors, radicicol or 17-allylaminodemethoxygeldanamycin (17-AAG), 24, 12, 6, and 0 hours before receiving a lethal dose of endotoxin (6.75 x 10(4) endotoxin units/g body weight). Outcomes included survival and parameters of systemic inflammation (plasma neutrophil, cytokine, chemokine, and nitrite/nitrate levels), pulmonary inflammation (lung nuclear factor-kappaB and myeloperoxidase activities, inducible nitric oxide synthase expression, inducible nitric oxide synthase-Hsp90 complex formation, and leukocyte infiltration), and lung injury (pulmonary capillary leak and lung function). MEASUREMENTS AND MAIN RESULTS: Mice pretreated with vehicle and receiving endotoxin exhibited 100% 24-hour lethality, a dramatic increase in all parameters of systemic and pulmonary inflammation, increased capillary leak, and reduced lung function. Compared with them, mice receiving either radicicol or 17-AAG before endotoxin exhibited prolonged survival, reduced or abolished increases in systemic and pulmonary inflammatory parameters, attenuated capillary leak, and restored, normal lung function. CONCLUSIONS: Hsp90 inhibitors may offer a new pharmacological tool in the management of severe sepsis and severe sepsis-induced ALI.

Sulforaphane suppresses angiogenesis and disrupts endothelial mitotic progression and microtubule polymerization.

Sulforaphane (SUL), an isothiocyanate derived from broccoli and other cruciferous vegetables, is known to induce phase II detoxification enzymes, disrupt cancer cell microtubule polymerization, and trigger cell cycle arrest in breast and colon cancer cells. Here, we provide the first evidence that SUL also acts to inhibit angiogenesis via suppression of endothelial cell proliferation. Bovine aortic endothelial (BAE) cells were exposed to concentrations of up to 15 microM SUL prior to cell cycle analysis and mitotic index quantification. Within 24 h, 15 microM SUL clearly induced G(2)/M accumulation and pre-metaphase arrest in BAE cells. Moreover, immunofluorescence tubulin staining indicated that this same SUL concentration was efficacious in not only disrupting mitotic progression, but also in perturbing normal polymerization of mitotic (and cytoplasmic) microtubules. Furthermore, daily administration of SUL (100 nmol/day, i.v. for 7 days) to female Balb/c mice bearing VEGF-impregnated Matrigel plugs strongly and significantly (P<0.05) suppressed angiogenesis progression as measured by hemoglobin concentration. Taken together, these findings suggest that the endothelial cell population is a novel target of SUL action both in vitro and in vivo. This mechanism of SUL-induced endothelial microtubule disruption and early mitotic arrest may further discern a potential role of SUL as a chemopreventive agent.