The dichotomous role of nitric oxide in the pathogenesis of accelerated atherosclerosis associated with systemic lupus erythematosus

Atherosclerosis is an inflammatory disorder, and the inflammation associated with systemic lupus erythematosus (SLE) accelerates the development of atherosclerosis. Nitric oxide (NO) is an important mediator of inflammation including the inflammation associated with atherosclerosis and SLE. Endothelial nitric oxide synthase (NOS3)-mediated constitutive expression of NO promotes endothelial integrity and normal vascular function. In contrast, inducible nitric oxide synthase- (NOS2) mediated expression of NO promotes endothelial dysfunction and atherogenesis. Statins appear to have anti-inflammatory properties and reverse many of the deleterious effects associated with NO metabolism in atherosclerosis. Statins augment NOS3 expression and inhibit the induction of NOS2. Therefore, the balance between normal vascular function and atherogenesis may be mediated by differences in the quantity, location, and timing of NO production within vessel walls.     

Nitric oxide and atherosclerosis.

Endothelial dysfunction has been shown in a wide range of vascular disorders including atherosclerosis and related diseases. Here, we examine and address the complex relationship among nitric oxide (NO)-mediated pathways and atherogenesis. In view of the numerous pathophysiological actions of NO, abnormalities could potentially occur at many sites: (a) impairment of membrane receptors in the arterial wall that interact with agonists or physiological stimuli capable of generating NO; (b) reduced concentrations or impaired utilization of l-arginine; (c) reduction in concentration or activity both of inducible and endothelial NO synthase; (d) impaired release of NO from the atherosclerotic damaged endothelium; (e) impaired NO diffusion from endothelium to vascular smooth muscle cells followed by decreased sensitivity to its vasodilator action; (f) local enhanced degradation of NO by increased generation of free radicals and/or oxidation-sensitive mechanisms; and (g) impaired interaction of NO with guanylate cyclase and consequent limitation of cyclic GMP production. Therefore, one target for new drugs should be the preservation or restoration of NO-mediated signaling pathways in arteries. Such novel therapeutic strategies may include administration of l-arginine/antioxidants and gene-transfer approaches.     

Phagocytosis is regulated by nitric oxide in murine microglia.

Nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) in activated microglia and has been shown to participate in host defense mechanisms. However, the role of NO produced by constitutive nitric oxide synthase (cNOS) in microglia is poorly understood. In this report, NO was found to regulate phagocytosis in murine BV-2 microglial cells as quantified by flow cytometry. Addition of NO-generating compounds caused impaired phagocytosis as compared to untreated microglia. The addition of nitric oxide synthase (NOS) inhibitors to microglial cells resulted in potentiation of phagocytosis, suggesting that constitutive NO was participating in the regulation of phagocytosis. The inverse correlation between NO production and phagocytosis was also observed when Alzheimer's beta-amyloid peptide was added. With beta-amyloid treatment, constitutive NO production decreased while phagocytosis increased. Cell extracts prepared from untreated microglia were found to contain both neuronal and endothelial NOS isoforms, but not the inducible form. The correlation of spontaneous NO production with attenuated phagocytosis suggests that constitutive NOS enzymes participate in microglial regulation.     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。     

Nitric oxide inhibition of homocysteine-induced human endothelial cell apoptosis by down-regulation of p53-dependent Noxa expression through the formation of S-nitrosohomocysteine

Hyperhomocysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; however, the pathogenic mechanism has not been clearly elucidated. In this study, we examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. Our data demonstrated that HCy caused caspase-dependent apoptosis in cultured human umbilical vein endothelial cells, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice but not in the p53-deficient cells. The NO donor S-nitroso-N-acetylpenicillamine, adenoviral transfer of inducible NO synthase gene, and antioxidants (alpha-tocopherol and superoxide dismutase plus catalase) but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by small interfering RNA-mediated suppression of Noxa expression, indicating that Noxa up-regulation plays an important role in HCy-induced endothelial cell apoptosis. Overexpression of inducible NO synthase increased the formation of S-nitroso-HCy, which was inhibited by the NO synthase inhibitor N-monomethyl-l-arginine. Moreover, S-nitroso-HCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.     

Presence and activity of nitric oxide synthase isoforms in ischemia-reperfusion-injured flaps

Nitric oxide is produced from the amino acid L-arginine by nitric oxide synthase, which has three known isoforms: (1) endothelial nitric oxide synthase and (2) brain nitric oxide synthase, both of which are constitutive nitric oxide synthase; and (3) inducible nitric oxide synthase. The authors' hypothesis is that after reperfusion injury, endothelial cell dysfunction leads to disruption of nitric oxide synthase-mediated nitric oxide production and that this may in part explain the deleterious effects of ischemia-reperfusion injury on tissue survival and blood reflow in flaps. An experiment was designed to study the effects of ischemia-reperfusion injury on the bioactivity of all three isoforms of nitric oxide synthase. Buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs. Flaps on one side of the animal were randomized to receive 6 hours of arterial ischemia, whereas flaps on the other side served as controls. At 6 hours of ischemia and at 1, 4, and 18 hours after reflow, tissue biopsy specimens were obtained and were processed for both constitutive nitric oxide synthase and inducible nitric oxide synthase enzyme activity on the basis of the L-citrulline assay. In addition, specimens were processed for Western blot analysis of the three isoforms. The authors' results revealed three key findings: first, there was a statistically significant (p < 0.001) decrease in constitutive nitric oxide synthase activity of ischemia-reperfusion-injured flaps as compared with controls in both skin and muscle for all time intervals measured. Second, Western blot analyses of endothelial nitric oxide synthase and brain nitric oxide synthase showed a significant decrease in the signal intensity in ischemic and reperfused tissue as compared with controls. Third, the inducible nitric oxide synthase isoform's activity and protein remained undetectable in both tissue types for all time points measured. The authors' data demonstrated that following ischemia-reperfusion injury in the pig flap model there was a disruption of constitutive nitric oxide synthase expression and activity, which may lead to decreased nitric oxide production. The significant decrease in nitric oxide synthase activity found in the current study may partly explain the mechanism of tissue damage in flaps subjected to ischemia-reperfusion injury. Knowledge of the kinetics of nitric oxide synthase activity under conditions of ischemia-reperfusion injury has important implications for the choice and timing of delivery of therapeutic agents whose goal is to increase the bioavailability of nitric oxide in reperfused tissue.     

Tetrahydrobiopterin attenuates homocysteine induced endothelial dysfunction

Homocysteine is an independent risk factor for atherosclerotic vascular disease. It impairs endothelial function via increasing superoxide production and quenching nitric oxide (NO) release. Tetrahydrobiopterin (BH₄) is a critical cofactor that couples nitric oxide synthase and facilitates the production of nitric oxide (vs. superoxide anions). In the first study, the effects of hyperhomocysteinemia (0.1 mM, 3 h) on endothelium-dependent vasorelaxation to ACh and A23187 were examined in isolated segments of rat aortae in the presence or absence of BH₄ (0.1 mM). In the second study, the effects of hyperhomocysteinemia (24 h) on nitric oxide production and superoxide release (using lucigenin chemiluminescence) were studied in human umbilical vein endothelial cells in the absence or presence of BH₄ (10 M). Homocysteine incubation impaired receptor-dependent and -independent endothelial function to ACh and A23187. This effect was attenuated by BH₄. Furthermore, homocysteine exposure increased superoxide production and impaired agonist-stimulated nitric oxide release. These effects were attenuated by BH₄ (p < 0.05). Hyperhomocysteinemia impairs endothelial function, in part due to a diminished bioavailability of BH₄ with resultant uncoupling of nitric oxide synthase. BH₄ may represent an important target for strategies aimed at improving endothelial dysfunction secondary to hyperhomocysteinemia.     

Molecular aspects of atherogenesis: new insights and unsolved questions

Summary The development of atherosclerotic disease results from the interaction between environment and genetic make up. A key factor in atherogenesis is the oxidative modification of lipids, which is involved in the recruitment of mononuclear leukocytes to the arterial intima – a process regulated by several groups of adhesion molecules and cytokines. Activated leukocytes, as well as endothelial mitochondria, can produce reactive oxygen species (ROS) that are associated with endothelial dysfunction, a cause of reduced nitric oxide (NO) bioactivity and further ROS production. Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) are nuclear receptors significantly involved in the control of lipid metabolism, inflammation and insulin sensitivity. Also, an emerging role has been suggested for G protein coupled receptors and for the small Ras and Rho GTPases in the regulation of the expression of endothelial NO synthase (eNOS) and of tissue factor, which are involved in thrombus formation and modulation of vascular tone. Further, the interactions among eNOS, cholesterol, oxidated LDL and caveola membranes are probably involved in some molecular changes observed in vascular diseases. Despite the relevance of oxidative processes in atherogenesis, anti-oxidants have failed to significantly improve atherosclerosis (ATS) prevention, while statins have proved to be the most successful drugs.     

Interleukin-1beta-induced nitric oxide production in rat aortic endothelial cells: inhibition by estradiol in normal and high glucose cultures.

Expression of inducible nitric oxide synthase (iNOS) and the resultant increased nitric oxide (NO) production are associated with septic shock, atherosclerosis, and cytokine-induced vascular injury. Estrogen is known to impact vascular injury and vascular tone, in part through regulation of NO production. In the current study, we examined the effect of physiological concentrations of estradiol on interleukin-1beta (IL-1beta)-induced NO production in rat aortic endothelial cells (RAECs). 17Beta-estradiol significantly decreased IL-1beta-induced iNOS protein levels and reduced NO production in RAECs. High glucose (25 mM) elevated the increase in IL-1beta-induced iNOS protein and NO production. Nevertheless, estradiol still inhibited IL-1beta-induced iNOS and NO production even in the presence of high glucose. These data suggest that estradiol may exert its beneficial effects in part by inhibiting induction of endothelial iNOS, a possible mechanism for the protective effect of estradiol against diabetes-associated cardiovascular complications.     

Role of Nitric Oxide in the Progression of Pneumoconiosis

Conflicting evidence has been reported as to whether nitric oxide (NO) possesses anti-inflammatory or inflammatory properties. Data are presented indicating that in vitro or in vivo exposure to selected occupational dusts, i.e., crystalline silica, organic dust contaminated with endotoxin, or asbestos, results in upregulation of inducible nitric oxide synthase (iNOS) and the production of NO by alveolar macrophages and pulmonary epithelial cells. Nitric oxide production is associated temporally and anatomically with pulmonary damage, inflammation, and disease progression in response to occupational dusts. Blockage of inducible nitric oxide synthase by administration of NOS inhibitors or in iNOS knockout mice decreases the magnitude of injury and inflammation following in vivo exposure to silica, endotoxin, or asbestos. Therefore, NO may play an important role in the initiation and progression of pneumoconiosis.     

The effects of nitric oxide on prostanoid production and release by human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) express and synthesize both constitutive and inducible nitric oxide synthase (NOS) and cyclo-oxygenase (COX) enzymes, and have been extensively used as an in vitro model to investigate the role of these enzymes in the patho-physiology of placenta-fetal circulation. In this study we investigated the role of NO in regulating prostanoid production and release from HUVEC. Both untreated and IL-1beta-treated HUVEC were exposed to various NOS inhibitors and NO donors in short-term (1 or 3 hours) experiments, and the effects on prostanoid production were evaluated through the measurement of prostaglandins (PG) I2, E2 and F2alpha released in the incubation medium. We found that the inhibition of inducible NOS but not endothelial NOS antagonizes the IL-1beta-induced increase in PGI2 release. However, NOS inhibitors do not modify baseline PGI2 production. Pharmacological levels of NO, obtained with various NO donors, inhibit basal and IL-1beta-stimulated PG release.     

S-Nitrosation of arginase 1 requires direct interaction with inducible nitric oxide synthase

Arginase constrains endothelial nitric oxide synthase activity by competing for the common substrate, L -Arginine. We have recently shown that inducible nitric oxide synthase (NOS2) S-nitrosates and activates arginase 1 (Arg1) leading to age-associated vascular dysfunction. Here, we demonstrate that a direct interaction of Arg1 with NOS2 is necessary for its S-nitrosation. The specific domain of NOS2 that mediates this interaction is identified. Disruption of this interaction in human aortic endothelial cells prevents Arg1 S-nitrosation and activation. Thus, disruption of NOS2-Arg1 interaction may represent a therapeutic strategy to attenuate age related vascular endothelial dysfunction.     

Vascular oxidative stress increases dendritic cell adhesion and transmigration induced by homocysteine

Atherosclerosis is a long-term chronic inflammatory and immunological disease. Endothelial dysfunction and the dendritic cell (DC) immune response are pivotal early events in atherogenesis. This study investigated the effects and possible mechanisms of action of homocysteine (Hcy) on DC adhesion to and transmigration between endothelial cells (ECs), and indicated a novel immunoregulatory mechanism by which Hcy induces atherogenesis. When ECs were stimulated with increasing concentrations of Hcy, immunofluorescence showed that endothelial reactive oxygen species (ROS) generation strikingly increased, while nitrite assay showed that nitric oxide (NO) release markedly decreased. Furthermore, DC adhesion and transmigration were significantly increased when ECs were activated by Hcy. However, pretreatment of ECs with antioxidant before Hcy markedly attenuated the induction of DC adhesion and transmigration, dependent on the intracellular ROS decrease and endothelial NO increase. In conclusion, DC adhesion and transmigration are significantly increased by vascular oxidative stress under conditions of elevated Hcy levels. These findings provide insight into the inflammatory processes and immune responses occurring in atherosclerosis induced by Hcy.     

Dehydroepiandrosterone stimulates nitric oxide release in vascular endothelial cells: evidence for a cell surface receptor

Dehydroepiandrosterone (DHEA) improves vascular function, but the mechanism of this effect is unclear. Since nitric oxide (NO) regulates vascular function, we hypothesized that DHEA affects the vasculature by increasing endothelial NO production. Physiological concentrations of DHEA stimulated NO release from intact bovine aortic endothelial cells (BAEC) within 5min. This effect was mediated by activation of endothelial nitric oxide synthase (eNOS) in BAEC and human umbilical vein endothelial cells (HUVEC). Dehydroepiandrosterone increased cyclic GMP (cGMP) levels in BAEC, consistent with its effect on NO production. Albumin-conjugated DHEA also stimulated NO release, suggesting that DHEA stimulates eNOS by a plasma membrane-initiated signal. Tamoxifen blocked estrogen-stimulated NO release from BAEC, but did not inhibit the DHEA effect. Pertussis toxin abolished the acute effect of DHEA on NO release. Dehydroepiandrosterone had no effect on intracellular calcium fluxes. However, inhibition of tyrosine kinases or the mitogen-activated protein (MAP) kinase kinase (MEK) blocked NO release and cGMP production in response to DHEA. These findings demonstrate that physiological concentrations of DHEA acutely increase NO release from intact vascular endothelial cells, by a plasma membrane-initiated mechanism. This action of DHEA is mediated by a steroid-specific, G-protein coupled receptor, which activates eNOS in both bovine and human cells. The release of NO is independent of intracellular calcium mobilization, but depends on tyrosine- and MAP kinases. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for DHEA.     

Nitric oxide paradox in asthma

Asthma results from allergen-driven intrapulmonary Th2 response, and is characterized by intermittent airway obstruction, airway hyperreactivity (AHR), and airway inflammation. Accumulating evidence indicates that inflammatory diseases of the respiratory tract are commonly associated with elevated production of nitric oxide (NO). It has been shown that exhaled NO may be derived from constitutive NO synthase (NOS) such as endothelial (NOS 3) and neural (NOS 1) in normal airways, while increased levels of NO in asthma appear to be derived from inducible NOS2 expressed in the inflamed airways. Nevertheless, the functional role of NO and NOS isoforms in the regulation of AHR and airway inflammation in human or experimental models of asthma is still highly controversial. In the present commentary we will discuss the role of lipopolysaccharides contamination of allergens as key element in the controversy related to the regulation of NOS2 activity in experimental asthma.     

Nitric oxide-dependent synthesis of vascular endothelial growth factor is impaired by high glucose

Synthesis of vascular endothelial growth factor (VEGF), the major angiogenic molecule, is induced by nitric oxide (NO) in various cell types, including vascular smooth muscle cells (VSMC). Therefore, compounds which inhibit NO generation can also influence VEGF synthesis. Here we investigated the effect of increased glucose concentration (25 mM vs. 5.5 mM) on cytokine-induced VEGF synthesis in rat VSMC. The cells growing in the medium containing 5.5 mM glucose and exposed to IL-1-beta, TNF-alpha and IFN-gamma induced expression of an inducible isoform of nitric oxide synthase (NOS II). This is followed by generation of NO and the concomitant expression of VEGF gene and release of VEGF protein. In contrast, 25 mM glucose impaired induction of NOS II expression and thus NO synthesis was lower than in 5.5 mM glucose. Consequently, the VEGF promoter activation was attenuated, resulting in decreased mRNA synthesis and lower production of VEGF protein. The results indicate that abnormally high concentrations of glucose can impair generation of NO and the NO-dependent VEGF synthesis. This may play a role in the development and progression of vascular dysfunctions in cardiovascular diseases.     

Role of heme oxygenase in preserving vascular bioactive NO

Beyond its vasodilator role, vascular nitric oxide (NO), which is synthesized by endothelial NO synthase (eNOS) via its activation, has been shown to play a number of other beneficial roles in the vascular system; it inhibits proliferation of vascular smooth muscle cells, prevents platelet aggregation, and regulates endothelial apoptosis. Such beneficial roles have been shown to be implicated in the regulation of endothelial functions. A loss of NO bioavailability that may result either from decreased eNOS expression and activity or from increased NO degradation is associated with endothelial dysfunction, a key factor in the development of vascular diseases. Heme oxygenase-1 (HO-1), an inducible enzyme, catalyzes the oxidative degradation of heme to free iron, carbon monoxide, and biliverdin, the latter being subsequently converted into bilirubin. In the vascular system, HO-1 and heme degradation products perform important physiological functions, which are ultimately linked to the protection of vascular cells. Studies have shown that HO-1 and heme degradation products exert vasodilatory, antioxidant, anti-inflammatory, antiproliferative and anti-apoptotic effects on vascular cells. Interestingly, these effects of HO-1 and its by-products are similar, at least in part, to those of eNOS-derived NO; this similarity may prompt investigators to study a possible relationship between eNOS-derived NO and HO-1 pathways. Many studies have been reported, and accumulating evidence suggests that HO-1 and heme degradation products can improve vascular function, at least in part, by compensating for the loss of NO bioavailability. This paper will provide the possible pathway explaining how HO-1 and heme degradation products can preserve vascular NO.     

Effects of nitric oxide synthase inhibitors on vascular hyperpermeability with thermal injury in mice.

The role of nitric oxide and related synthase in thermal injury was investigated by using models of experimental burn to evaluate severity from the aspect of vascular permeability. Thermal injuries were produced in the murine right ear by pinching with a pair of preheated tweezers. Immediately thereafter, Evans blue dye was intravenously administered, and the mice injured with burns were sacrificed at various times. The burned ears were collected and hydrolyzed, and the level of extracted dye was measured as an indicator of inflammation. Vascular hyperpermeability was suppressed by the administration of nitric oxide synthase inhibitors. LNAME not only suppressed vascular hyperpermeability in thermal injuries in a dose-dependent manner but was also effective with either prophylactic or therapeutic administration. Although aminoguanidine also suppressed the inflammatory response, it had no effect on the early inflammatory phase. Nitric oxide synthase is well known to have two types of isozymes. Aminoguanidine, an inhibitor specific to inducible nitric oxide synthase, suppressed the late phase 6 h after injury, suggesting that inducible nitric oxide synthase is involved in inflammatory responses of thermal injuries. These results also demonstrated that inducible nitric oxide synthase-like protein stained the burned region immunohistochemically. Therefore, both types of enzymes mediating nitric oxide affect inflammatory responses, i.e., vascular hyperpermeability, and their regulation may lead to the development of new therapy for thermal injuries.