Endothelial Nitric Oxide Synthase Knockout Mice Research Articles

Augmentation of Nitric Oxide Deficient Hypertension by High Salt Diet Is Associated With Reduced TNF-α Receptor Type 1 Expression in the Kidneys.

High salt (HS) intake induces an augmented hypertensive response to nitric oxide (NO) inhibition, though it causes minimal changes in blood pressure (BP) in NO intact condition. The cause of such augmentation is not known. HS induces tumor necrosis factor-alpha (TNFα) production that causes natriuresis via activation of its receptor type 1 (TNFR1). We hypothesized that NO deficiency reduces renal TNFR1 activity, leading to enhanced sodium retention and hypertension. We examined the changes in renal TNFR1 protein expression (Immunohistochemistry analyses) after HS (4% NaCl) intake in wild-type mice (WT, C57BL6) treated with a NO synthase (NOS) inhibitor, nitro-l-arginine methyl ester (L-NAME; 0.05 mg/min/g; osmotic mini-pump), as well as in endothelial NOS knockout mice (eNOSKO) and compared the responses in WT mice with normal salt (NS; 0.3% NaCl) intake. BP was measured with tail-cuff plethysmography and 24-hour urine collections were made using metabolic cages. HS alone did not alter mean BP in untreated mice (76 ± 3 to 77 ± 1 mm Hg) but induced an augmented response in L-NAME treated (106 ± 1 vs. 97 ± 2 mm Hg) and in eNOSKO (107 ± 2 vs. 89 ± 3 mm Hg) mice. The percentage area of TNFR1 expression in renal tissue was higher in WT + HS (4.1 + 0.5%) than in WT + NS mice (2.7 ± 0.6%). However, TNFR1 expression was significantly lower in L-NAME treated WT + NS (0.9 ± 0.1%) and in eNOSKO + NS (1.4 ± 0.2%) than in both WT + NS and WT + HS mice. These data indicate that TNFR1 activity is downregulated in NO deficient conditions, which facilitates salt retention leading to augmented hypertension during HS intake.

Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically relevant metabolic enzymes

Cysteine S-nitrosation is a reversible post-translational modification mediated by nitric oxide (•NO)-derived agents. S-Nitrosation participates in cellular signaling and is associated with several diseases such as cancer, cardiovascular diseases, and neuronal disorders. Despite the physiological importance of this nonclassical •NO-signaling pathway, little is understood about how much S-nitrosation affects protein function. Moreover, identifying physiologically relevant targets of S-nitrosation is difficult because of the dynamics of transnitrosation and a limited understanding of the physiological mechanisms leading to selective protein S-nitrosation. To identify proteins whose activities are modulated by S-nitrosation, we performed a metabolomics study comparing WT and endothelial nitric-oxide synthase knockout mice. We integrated our results with those of a previous proteomics study that identified physiologically relevant S-nitrosated cysteines, and we found that the activity of at least 21 metabolic enzymes might be regulated by S-nitrosation. We cloned, expressed, and purified four of these enzymes and observed that S-nitrosation inhibits the metabolic enzymes 6-phosphogluconate dehydrogenase, Δ1-pyrroline-5-carboxylate dehydrogenase, catechol-O-methyltransferase, and d-3-phosphoglycerate dehydrogenase. Furthermore, using site-directed mutagenesis, we identified the predominant cysteine residue influencing the observed activity changes in each enzyme. In summary, using an integrated metabolomics approach, we have identified several physiologically relevant S-nitrosation targets, including metabolic enzymes, which are inhibited by this modification, and we have found the cysteines modified by S-nitrosation in each enzyme.

Glucagon-Like Peptide 2 (GLP-2) Stimulates Postprandial Chylomicron Production and Postabsorptive Release of Intestinal Triglyceride Storage Pools via Induction of Nitric Oxide Signaling in Male Hamsters and Mice.

The intestinal overproduction of apolipoprotein B48 (apoB48)-containing chylomicron particles is a common feature of diabetic dyslipidemia and contributes to cardiovascular risk in insulin resistant states. We previously reported that glucagon-like peptide-2 (GLP-2) is a key endocrine stimulator of enterocyte fat absorption and chylomicron output in the postprandial state. GLP-2's stimulatory effect on chylomicron production in the postabsorptive state has been confirmed in human studies. The mechanism by which GLP-2 regulates chylomicron production is unclear, because its receptor is not expressed on enterocytes. We provide evidence for a key role of nitric oxide (NO) in mediating the stimulatory effects of GLP-2 during the postprandial and postabsorptive periods. Intestinal chylomicron production was assessed in GLP-2-treated hamsters administered the pan-specific NO synthase (NOS) inhibitor L-N(G)-nitroarginine methyl ester (L-NAME), and in GLP-2-treated endothelial NOS knockout mice. L-NAME blocked GLP-2-stimulated apoB48 secretion and reduced triglycerides (TGs) in the TG-rich lipoprotein (TRL) fraction of the plasma in the postprandial state. Endothelial NOS-deficient mice were resistant to GLP-2 stimulation and secreted fewer large apoB48-particles. When TG storage pools were allowed to accumulate, L-NAME mitigated the GLP-2-mediated increase in TRL-TG, suggesting that NO is required for early mobilization and secretion of stored TG and preformed chylomicrons. Importantly, the NO donor S-nitroso-L-glutathione was able to elicit an increase in TRL-TG in vivo and stimulate chylomicron release in vitro in primary enterocytes. We describe a novel role for GLP-2-mediated NO-signaling as a critical regulator of intestinal lipid handling and a potential contributor to postprandial dyslipidemia.

Stem cell factor is a potent endothelial permeability factor.

Although stem cell factor (SCF) has been shown to play a critical role in hematopoiesis, gametogenesis, and melanogenesis, the function of SCF in the regulation of vascular integrity has not been studied. We demonstrated that SCF binds to and activates the cKit receptor in endothelial cells, thereby increasing the internalization of vascular endothelial-cadherin and enhancing extravasation of dyes to a similar extent as vascular endothelial growth factor. SCF-mediated cKit activation in endothelial cells enhanced the phosphorylation of endothelial nitric oxide (NO) synthase via the phosphoinositide 3-kinase/Akt signaling pathway and subsequently increased the production of NO. Inhibition of endothelial NO synthase expression and NO synthesis using small interfering RNA knockdown and chemical inhibitors substantially diminished the ability of SCF to increase the internalization of vascular endothelial-cadherin and in vitro endothelial permeability. SCF-induced increase in extravasation of the dyes was abrogated in endothelial NO synthase knockout mice, which indicates that endothelial NO synthase-mediated NO production was responsible for the SCF-induced vascular leakage. Furthermore, we demonstrated that the expression of SCF and cKit was significantly higher in the retina of streptozotocin-injected diabetic mice than in the nondiabetic control animals. Depletion of SCF by intravitreous injection of anti-SCF-neutralizing immunoglobulin G significantly prevented vascular hyperpermeability in the retinas of streptozotocin-injected diabetic mice. Our data reveal that SCF disrupts the endothelial adherens junction and enhances vascular leakage, as well as suggest that anti-SCF/cKit therapy may hold promise as a potential therapy for the treatment of hyperpermeable vascular diseases.

Adiponectin‐Mediated Modulation of Lymphatic Vessel Formation and Lymphedema

BackgroundObesity is linked with an increased risk of lymphedema, which is a serious clinical problem. Adiponectin is a circulating adipokine that is down‐regulated in obese states. We investigated the effects of adiponectin on lymphatic vessel formation in a model of lymphedema and dissected its mechanisms.Methods and ResultsA mouse model of lymphedema was created via ablation of tail surface lymphatic network. Adiponectin‐knockout mice showed the greater diameter of the injured tail compared with wild‐type mice, which was associated with lower numbers of lymphatic endothelial cells (LECs). Systemic delivery of adiponectin reduced the thickness of the injured tail and enhanced LEC formation in wild‐type and adiponectin‐knockout mice. Adiponectin administration also improved the edema of injured tails in obese KKAy mice. Treatment with adiponectin protein stimulated the differentiation of human LECs into tubelike structures and increased LEC viability. Adiponectin treatment promoted the phosphorylation of AMP‐activated protein kinase (AMPK), Akt, and endothelial nitric oxide synthase n LECs. Blockade of AMPK or Akt activity abolished adiponectin‐stimulated increase in LEC differentiation and viability and endothelial nitric oxide synthase phosphorylation. Inhibition of AMPK activation also suppressed adiponectin‐induced Akt phosphorylation in LECs. In contrast, inactivation of Akt signaling had no effects on adiponectin‐mediated AMPK phosphorylation in LECs. Furthermore, adiponectin administration did not affect the thickening of the damaged tail in endothelial nitric oxide synthase–knockout mice.ConclusionsAdiponectin can promote lymphatic vessel formation via activation of AMPK/Akt/endothelial nitric oxide synthase signaling within LECs, thereby leading to amelioration of lymphedema.

Hydrogen Sulfide Stimulates Ischemic Vascular Remodeling Through Nitric Oxide Synthase and Nitrite Reduction Activity Regulating Hypoxia‐Inducible Factor‐1α and Vascular Endothelial Growth Factor–Dependent Angiogenesis

BackgroundHydrogen sulfide (H2S) therapy is recognized as a modulator of vascular function during tissue ischemia with the notion of potential interactions of nitric oxide (NO) metabolism. However, little is known about specific biochemical mechanisms or the importance of H2S activation of NO metabolism during ischemic tissue vascular remodeling. The goal of this study was to determine the effect of H2S on NO metabolism during chronic tissue ischemia and subsequent effects on ischemic vascular remodeling responses.Methods and ResultsThe unilateral, permanent femoral artery ligation model of hind‐limb ischemia was performed in C57BL/6J wild‐type and endothelial NO synthase–knockout mice to evaluate exogenous H2S effects on NO bioavailability and ischemic revascularization. We found that H2S selectively restored chronic ischemic tissue function and viability by enhancing NO production involving both endothelial NO synthase and nitrite reduction mechanisms. Importantly, H2S increased ischemic tissue xanthine oxidase activity, hind‐limb blood flow, and angiogenesis, which were blunted by the xanthine oxidase inhibitor febuxostat. H2S treatment increased ischemic tissue and endothelial cell hypoxia‐inducible factor‐1α expression and activity and vascular endothelial growth factor protein expression and function in a NO‐dependent manner that was required for ischemic vascular remodeling.ConclusionsThese data demonstrate that H2S differentially regulates NO metabolism during chronic tissue ischemia, highlighting novel biochemical pathways to increase NO bioavailability for ischemic vascular remodeling.

Roles of CYP2C29 and RXRγ in vascular EET synthesis of female mice

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

Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice

High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V(T). Ventilation with 20 ml/kg V(T) for 80 min increased the filtration coefficient (K(f)), an index of vascular permeability. The increased cGMP and K(f) caused by HV(T) were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 muM ODQ) also blocked cGMP generation and inhibited the increase in K(f), suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wet-to-dry weight ratio in an anesthetized mouse model of HV(T) injury. Additional activation of sGC (1.5 muM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K(f) in lungs ventilated with 15 ml/kg V(T). HV(T) endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 muM IBMX) as well as an inhibitor (10 muM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V(T)-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV(T)-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.

Neuronal Nitric Oxide Synthase Contributes to the Regulation of Hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopoiesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells.

Increased Salt-Sensitivity in Endothelial Nitric Oxide Synthase–Knockout Mice

Although impaired nitric oxide production contributes importantly to salt-sensitivity, the role of the endothelial isoform of nitric oxide synthase (eNOS) has received little attention. In the present study we compared the effects of a high-salt diet on the blood pressure response of eNOS knockout (eNOS-/-) and control (eNOS+/+) mice. Mean arterial pressure (MAP), heart rate, pulse pressure, and activity levels were recorded by telemetry in mice fed a regular-salt diet (0.7% NaCl) followed by 6 weeks on either a high-salt (8% NaCl) or regular-salt diet. The eNOS-/- mice exhibited a 15% increase in MAP and a 2- to 2.5-fold increase in salt-sensitivity relative to the control strain. Salt-induced increases in MAP were well sustained in eNOS-/-, whereas in eNOS+/+ the initial increase was biphasic. The effects of salt on MAP were particularly pronounced during locomotor activity, during the dark phase, and at the peak levels of MAP recorded over the course of the day. The high-salt diet also led to a transient increase in the proportion of time spent active. Levels of heart rate and pulse pressure were relatively unaffected by the high-salt diet. The eNOS-/- mice exhibit an increased blood pressure response to a high-salt diet. This finding suggests that eNOS normally provides an important contribution to the body's adaptation to a salt load and that reduced production of NO by eNOS may promote salt-sensitivity and salt-induced hypertension.

Arginase: a modulator of myocardial function

when nitric oxide (NO) donors were first administered to isolated, normal cardiomyocytes, a negative, cGMP-dependent inotropic effect was observed, whereas inhibitors of NO synthases (NOS) had no effect. Further studies then demonstrated a biphasic contractile response to exogenous NO and cGMP,

The effects of arg-vasopressin on osteoblast-like cells in endothelial nitric oxide synthase-knockout mice and their wild type counterparts

In the present study, we investigated whether nitric oxide (NO) could be involved in the effects of arg-vasopressin (AVP) on osteoblast-like cells. Cells derived from endothelial nitric oxide synthase (eNOS)-knockout mice and their wild type (WT) counterparts, and an osteosarcoma cell line (SaOS-2) were used. AVP (10–100 pmol/l) increased proliferation of osteoblast-like cells from WT mice. The effect was abolished by an AVP V1-receptor antagonist. AVP increased proliferation of cells from eNOSKO mice only when a NO donor, SNAP, was added. A nitric oxide synthase-inhibitor, L-NAME, antagonized the increase in cell proliferation in response to AVP in SaOS-2 cells. In conclusion, this study indicates that NO is involved in the effects of AVP on cell proliferation in osteoblast-like cells.

Enhanced or Repressed Pressor Responses to Endothelin-1 or IRL-1620, respectively, in eNOS Knockout Mice

Nitric oxide (NO) has been shown to be a physiological antagonist of the potent pressor agent endothelin-1, a transcriptional and post-transcriptional repressor of the production of this particular peptide as well as an endogenous modulator of vascular resistance in several animal species as well as in man. The aim of the present study was to assess the hemodynamic properties of endothelin-1 in endothelial NO synthase knockout mice (eNOS (-/-) KO). Ketamine- xylazine anesthetized eNOS (-/-) KO mice show a significantly higher mean arterial pressure (+25.7 +/- 4.3 mmHg, n = 10) than their wild-type congeners (C57BL/6 background). eNOS (-/-) KO mice, when subjected to systemically administered endothelin-1 or the selective endothelin-B agonist IRL-1620 (1 nmol/kg, each), responded by markedly enhanced and reduced pressor responses (systolic, diastolic and mean arterial pressure), respectively, when compared with wild-type mice. Surprisingly, plasma levels of immunoreactive endothelin are not significantly different in endothelial nitric oxide synthase knockout mice and wild-type mice. Our results suggest that chronic repression of endothelial nitric oxide synthase affects in opposite fashion the functionality of endothelin-A and endothelin-B receptor-dependent responses. The increase in mean arterial pressure afforded in endothelial nitric oxide synthase knockout mice does not appear to involve an increase in production of endogenous endothelin in vivo.

Inhibited anabolic effect of insulin-like growth factor-I on stromal bone marrow cells in endothelial nitric oxide synthase-knockout mice.

Insulin-like growth factor-I (IGF-I), parathyroid hormone (PTH) and PTH-related protein (PTHrP) are hormones that have anabolic effects on bone formation. The aim of this study was to investigate whether production of nitric oxide (NO) is involved in the effect of IGF-I and PTH/PTHrP on osteoblast-like cells. Bone marrow stromal cells from adult endothelial nitric oxide synthase (eNOS)-knockout (eNOSKO) mice and wild type (WT) counterparts were cultivated with osteogenic substances. The cells showed an osteoblastic phenotype measured as osteocalcin production and alkaline phosphatase activity. DNA synthesis was measured as [3H] thymidine incorporation in the bone marrow cells and in a human osteosarcoma cell-line (SaOS-2). The stimulatory effect of IGF-I on thymidine incorporation seen in WT animals was absent in eNOSKO mice. Addition of a NO donor to eNOSKO cells recovered the effect of IGF-I on thymidine incorporation. PTH/PTHrP stimulated cell proliferation in both WT and eNOSKO mice. In SaOS-2 cells, incubation with IGF-I together with a NOS inhibitor resulted in an inhibition of the anabolic effect of IGF-I on cell proliferation. The stimulatory effect of IGF-I on WT cell proliferation was abolished in eNOSKO cells, recovered by an NO donor and inhibited in osteosarcoma cells by a NOS inhibitor. The results indicate that the effect of IGF-I is dependent on NO production. The impaired IGF-I response may contribute to the bone defect formation seen in eNOSKO animals.

An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice.

In addition to nitric oxide (NO) and prostacyclin (PGI(2)), the endothelium generates the endothelium-derived hyperpolarizing factor (EDHF). We set out to determine whether an EDHF-like response can be detected in wild-type (WT) and endothelial NO synthase knockout mice (eNOS -/-) mice. Vasodilator responses to endothelium-dependent agonists were determined in vivo and in vitro. In vivo, bradykinin induced a pronounced, dose-dependent decrease in mean arterial pressure (MAP) which did not differ between WT and eNOS -/- mice and was unaffected by treatment with N(omega)-nitro-l-arginine methyl ester and diclofenac. In the saline-perfused hindlimb of WT and eNOS -/- mice, marked N(omega)-nitro-l-arginine (l-NA, 300 micromol/liter)- and diclofenac-insensitive vasodilations in response to both bradykinin and acetylcholine (ACh) were observed, which were more pronounced than the agonist-induced vasodilation in the hindlimb of WT in the absence of l-NA. This endothelium-dependent, NO/PGI(2)-independent vasodilatation was sensitive to KCl (40 mM) and to the combination of apamin and charybdotoxin. Gap junction inhibitors (18alpha-glycyrrhetinic acid, octanol, heptanol) and CB-1 cannabinoid-receptor agonists (Delta(9)-tetrahydrocannabinol, HU210) impaired EDHF-mediated vasodilation, whereas inhibition of cytochrome P450 enzymes, soluble guanylyl cyclase, or adenosine receptors had no effect on EDHF-mediated responses. These results demonstrate that in murine resistance vessels the predominant agonist-induced endothelium-dependent vasodilation in vivo and in vitro is not mediated by NO, PGI(2), or a cytochrome P450 metabolite, but by an EDHF-like principle that requires functional gap junctions.