Isoprenoid depletion by statins antagonizes cytokine-induced down-regulation of endothelial nitric oxide expression and increases NO synthase activity in human umbilical vein endothelial cells.

Endothelial dysfunction and atherosclerosis are associated with an inflammation-induced decrease in endothelial nitric oxide synthase (eNOS) expression. Based on the differences between hydrophobic and hydrophilic statins in their reduction of cardiac events, we analyzed the effects of rosuvastatin and cerivastatin on eNOS and inducible NO synthase (iNOS) expression and NOS activity in TNF-alpha-stimulated human umbilical vein endothelial cells (HUVEC). Both statins reversed down-regulation of eNOS mRNA and protein expression by inhibiting HMG-CoA reductase and isoprenoid synthesis. Cerivastatin tended to a more pronounced effect on eNOS expression compared to rosuvastatin. NOS activity - measured by conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline - was enhanced under treatment with both drugs due to inhibition of HMG-CoA reductase. Statin-treatment reduced iNOS mRNA expression under normal conditions, but had no relevant effects on iNOS mRNA expression in cytokine-treated cells. Rosuvastatin and cerivastatin reverse the detrimental effects of TNF-alpha-induced down-regulation in eNOS protein expression and increase NO synthase activity by inhibiting HMG-CoA reductase and subsequent blocking of isoprenoid synthesis. These results provide evidence that statins have beneficial effects by increasing eNOS expression and activity during the atherosclerotic process.     

Reduction of blood pressure,plasma cholesterol,and atherosclerosis by elevated endothelial nitric oxide

In the vascular system, nitric oxide is generated by endothelial NO synthase (eNOS). NO has pleiotropic effects, most of which are believed to be atheroprotective. Therefore, it has been argued that patients suffering from cardiovascular disease could benefit from an increase in eNOS activity. However, increased NO production can cause oxidative damage, cell toxicity, and apoptosis and hence could be atherogenic rather than beneficial. To study the in vivo effects of increased eNOS activity, we created transgenic mice overexpressing human eNOS. Aortic blood pressure was approximately 20 mm Hg lower in the transgenic mice compared with control mice because of lower systemic vascular resistance. The effects of eNOS overexpression on diet-induced atherosclerosis were studied in apolipoprotein E-deficient mice. Elevation of eNOS activity decreased blood pressure ( approximately 20 mm Hg) and plasma levels of cholesterol ( approximately 17%), resulting in a reduction in atherosclerotic lesions by 40%. We conclude that an increase in eNOS activity is beneficial and provides protection against atherosclerosis.     

Pathogenetic role of eNOS uncoupling in cardiopulmonary disorders

The homodimeric flavohemeprotein endothelial nitric oxide synthase (eNOS) oxidizes l-arginine to l-citrulline and nitric oxide (NO), which acutely vasodilates blood vessels and inhibits platelet aggregation. Chronically, eNOS has a major role in the regulation of blood pressure and prevention of atherosclerosis by decreasing leukocyte adhesion and smooth muscle proliferation. However, a disturbed vascular redox balance results in eNOS damage and uncoupling of oxygen activation from l-arginine conversion. Uncoupled eNOS monomerizes and generates reactive oxygen species (ROS) rather than NO. Indeed, eNOS uncoupling has been suggested as one of the main pathomechanisms in a broad range of cardiovascular and pulmonary disorders such as atherosclerosis, ventricular remodeling, and pulmonary hypertension. Therefore, modulating uncoupled eNOS, in particular eNOS-dependent ROS generation, is an attractive therapeutic approach to preventing and/or treating cardiopulmonary disorders, including protective effects during cardiothoracic surgery. This review provides a comprehensive overview of the pathogenetic role of uncoupled eNOS in both cardiovascular and pulmonary disorders. In addition, the related therapeutic possibilities such as supplementation with the eNOS substrate l-arginine, volatile NO, and direct NO donors as well as eNOS modulators such as the eNOS cofactor tetrahydrobiopterin and folic acid are discussed in detail.     

Vaspin Increases Nitric Oxide Bioavailability through the Reduction of Asymmetric Dimethylarginine in Vascular Endothelial Cells

Vaspin is an adipocytokine recently identified in the visceral adipose tissue of diabetic rats and having anti-diabetic effects. We have recently shown that vaspin has anti-atherogenic effect through Akt-mediated inhibition of endothelial cell apoptosis. Decreased activity of endothelial nitric oxide synthase (eNOS) plays an important role in the pathogenesis of atherosclerosis. Asymmetric dimethylarginine (ADMA) is a well-known endogenous competitive inhibitor of eNOS and risk factor of cardiovascular diseases. The aim of this study was to examine whether vaspin might protect against atherosclerosis through its beneficial effects on the ADMA-eNOS system. Treatment of vaspin significantly increased NO secretion from endothelial cells and isolated aorta from Sprague-Dawley (SD) rats. Furthermore, treatment of vaspin prevented fatty acid-induced decrease in endothelium-dependent vasorelaxation in isolated aorta of SD rat. For the mechanism of vaspin-induced NO biosynthesis, vaspin activated the STAT3 signaling pathway and stimulated eNOS phosphorylation (Ser 1177), a marker of eNOS activation, through STAT3-dependent mechanism. Furthermore, vaspin treatment increased the expression of dimethylarginine dimethylaminohydrolase (DDAH) II, the responsible enzyme for the degradation of ADMA, leading to a reduction in ADMA levels. Vaspin-induced increase in DDAH II gene expression was through STAT3-mediated stimulation of DDAH II promoter activity. These results suggest that vaspin increases eNOS activity by reducing ADMA level through STAT3-mediated regulation of DDAH II expression. Our findings provide a novel molecular mechanism of antiatherogenic actions of vaspin.     

Oxidized LDL at low concentration promotes in-vitro angiogenesis and activates nitric oxide synthase through PI3K/Akt/eNOS pathway in human coronary artery endothelial cells

It has long been considered that oxidized low-density lipoprotein (oxLDL) causes endothelial dysfunction and is remarkably related to the development of atherosclerosis. However, the effect of oxLDL at very low concentration (<10 μg/ml) on the endothelial cells remains speculative. Nitric oxide (NO) has a crucial role in the endothelial cell function. In this study, we investigated the effect of oxLDL at low concentration on NO production and proliferation, migration, tube formation of the human coronary artery endothelial cells (HCAEC). Results showed that oxLDL at 5 μg/ml enhanced HCAEC proliferation, migration and tube formation. These phenomena were accompanied by an increased intracellular NO production. l-NAME (a NOS inhibitor), LY294002 and wortmannin (PI3K inhibitors) could abolish oxLDL-induced angiogenic effects and prevent NO production in the HCAEC. The phosphorylation of Akt, PI3K and eNOS were up-regulated by oxLDL, which was attenuated by LY294002. Our results suggested that oxLDL at low concentration could promote in-vitro angiogenesis and activate nitric oxide synthesis through PI3K/Akt/eNOS pathway in HCAEC.     

Regulation of endothelial-type NO synthase expression in pathophysiology and in response to drugs.

In many types of cardiovascular pathophysiology such as hypercholesterolemia and atherosclerosis, diabetes, cigarette smoking, or hypertension (with its sequelae stroke and heart failure) the expression of endothelial NO synthase (eNOS) is altered. Both up- and downregulation of eNOS have been observed, depending on the underlying disease. When eNOS is upregulated, the upregulation is often futile and goes along with a reduction in bioactive NO. This is due to an increased production of superoxide generated by NAD(P)H oxidase and by an uncoupled eNOS. A number of drugs with favorable effects on cardiovascular disease upregulate eNOS expression. The resulting increase in vascular NO production may contribute to their beneficial effects. These compounds include statins, angiotensin-converting enzyme inhibitors, AT1 receptor antagonists, calcium channel blockers, and some antioxidants. Other drugs such as glucocorticoids, whose administration is associated with cardiovascular side effects, downregulate eNOS expression. Stills others such as the immunosuppressants cyclosporine A and FK506/tacrolimus or erythropoietin have inconsistent effects on eNOS. Thus regulation of eNOS expression and activity contributes to the overall action of several classes of drugs, and the development of compounds that specifically upregulate this protective enzyme appears as a desirable target for drug development.     

Stat3 mediates interleukin-6 [correction of interelukin-6] inhibition of human endothelial nitric-oxide synthase expression

Chronic activation of the acute phase response (APR) is associated with atherosclerosis. Elevated levels of interleukin-6, the major inducer of the APR, are associated with an increased risk of cardiovascular events. One of the clinical hallmarks of atherogenesis is endothelial dysfunction, characterized by a decrease in endothelial production of nitric oxide (NO). We hypothesized that interleukin-6 (IL-6) decreases endothelial NO synthase (eNOS) expression. We now show that IL-6 treatment of human aortic endothelial cells (HAEC) decreases steady-state levels of human eNOS mRNA and protein. This decrease in eNOS expression is caused in part by IL-6 inhibition of transactivation of the human eNOS promoter. To explore the mechanism by which IL-6 affects eNOS expression, we examined activation of signal transducer and transactivator-3 (Stat3). The IL-6 receptor (IL-6R) is expressed in HAEC, and Stat3 is phosphorylated in response to IL-6 stimulation of the IL-6R. We identified four consensus sequences for Stat3 binding (SIE) in the eNOS promoter at positions -1520, -1024, -840, and -540. Transfection of eNOS promoter mutants revealed that the SIE at -1024 mediates Stat3 inhibition of eNOS promoter activity. Gel-shift analysis of nuclear extracts from HAEC treated with IL-6 confirms that Stat3 binds to a complex containing the SIE at -1024. RNA silencing of STAT3 blocks the inhibitory effect of IL-6 on eNOS expression. Our data show that IL-6 has direct effects upon endothelial cells, inhibiting eNOS expression in part through Stat3. Decreased levels of eNOS may be an important component of the pro-atherogenic effect of the APR.     

Superoxide production and reactive oxygen species signaling by endothelial nitric-oxide synthase

Reactive oxygen species can function as intracellular messengers, but linking these signaling events with specific enzymes has been difficult. Purified endothelial nitric-oxide synthase (eNOS) can generate superoxide (O(2)) under special conditions but is only known to participate in cell signaling through NO. Here we show that eNOS regulates tumor necrosis factor alpha (TNFalpha) through a mechanism dependent on the production of O(2) and completely independent of NO. Expression of eNOS in transfected U937 cells increased phorbol 12-myristate 13-acetate-induced TNFalpha promoter activity and TNFalpha production. N(omega)-Methyl-l-arginine, an inhibitor of eNOS that blocks NO production but not its NADPH oxidase activity, did not prevent TNFalpha up-regulation. Likewise, Gln(361)eNOS, a competent NADPH oxidase that lacks NOS activity, retained the ability to increase TNFalpha. Similar to the effect of eNOS, a O(2) donor dose-dependently increased TNFalpha production in differentiated U937 cells. In contrast, cotransfection of superoxide dismutase with eNOS prevented TNFalpha up-regulation, as did partial deletion of the eNOS NADPH binding site, a mutation associated with loss of O(2) production. Thus, eNOS may straddle a bifurcating pathway that can lead to the formation of either NO or O(2), interrelated but often opposing free radical messengers. This arrangement has possible implications for atherosclerosis and septic shock where endothelial dysfunction results from imbalances in NO and O(2) production.     

Effects of oxidized low density lipoprotein on nitric oxide synthetase and protein kinase C activities in bovine endothelial cells.

Oxidized low-density lipoprotein (Ox-LDL) is an atherogenic lipoprotein. It has been suggested that Ox-LDL causes endothelial dysfunction by decreasing the release of endothelium-derived factors (EDRF-NO) or increasing the inactivation of EDRF-NO. The mechanism by which Ox-LDL causes dysfunctional NO during early stages of atherosclerosis is not clear. The purpose of this study was to examine the role of Ox-LDL on nitric oxide synthetase (eNOS), protein kinase C (PKC) activities and cAMP production in bovine aortic endothelial cells (BAEC). Ox-LDL stimulated PKC activity of BAEC but it inhibited both eNOS activity and cAMP production. Ox-LDL partially inhibited the forskolin stimulated cAMP production. Furthermore, we observed that 8Br-cAMP treatment decreased the activity of eNOS in a concentration dependent manner. Serotonin which has a profound inhibitory effect on cAMP production also stimulated eNOS activity. Pertusis toxin treatment blocked the stimulatory action of serotonin on the stimulation of eNOS activity. Our results thus suggest that Ox-LDL inhibit the endothelium-dependent relaxation. One possible mechanism is that Ox-LDL stimulates PKC activity, which in turn increases the phosphorylation of the Gi-protein. Inhibition of Gi-protein then leads to reduced release of NO from endothelial cells and thus causes endothelial dysfunction.     

Endothelin-1 activates endothelial cell nitric-oxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein jinase B/Akt

Endothelin-1 has dual vasoactive effects, mediating vasoconstriction via ETA receptor activation of vascular smooth muscle cells and vasorelaxation via ETB receptor activation of endothelial cells. Although it is commonly accepted that endothelin-1 binding to endothelial cell ETB receptors stimulates nitric oxide (NO) synthesis and subsequent smooth muscle relaxation, the signaling pathways downstream of ETB receptor activation are unknown. Here, using a model in which we have utilized isolated primary endothelial cells, we demonstrate that ET-1 binding to sinusoidal endothelial cell ETB receptors led to increased protein kinase B/Akt phosphorylation, endothelial cell nitric-oxide synthase (eNOS) phosphorylation, and NO synthesis. Furthermore, eNOS activation was not dependent on tyrosine phosphorylation, and pretreatment of endothelial cells with pertussis toxin as well as overexpression of a dominant negative G-protein-coupled receptor kinase construct that sequesters betagamma subunits inhibited Akt phosphorylation and NO synthesis. Taken together, the data elucidate a G-protein-coupled receptor signaling pathway for ETB receptor-mediated NO production and call attention to the absolute requirement for heterotrimeric G-protein betagamma subunits in this cascade.     

Endothelial NADH/NADPH-dependent enzymatic sources of superoxide production: relationship to endothelial dysfunction

There is growing evidence that endothelial dysfunction, which is often defined as the decreased endothelial-derived nitric oxide (NO) bioavailability, is a crucial factor leading to vascular disease states such as hypertension, diabetes, atherosclerosis, heart failure and cigarette smoking. This is due to the fact that the lack of NO in endothelium-dependent vascular disorders contributes to impaired vascular relaxation, platelet aggregation, increased vascular smooth muscle proliferation, and enhanced leukocyte adhesion to the endothelium. During the last several years, it has become clear that reduction of NO bioavailability in the endothelium-impaired function disorders is associated with an increase in endothelial production of superoxide (O(2)(*-)). Because O(2)(*-) rapidly scavenges NO within the endothelium, a reduction of bioactive NO might occur despite an increased NO generation. Among many enzymatic systems that are capable of producing O(2)(*-), NAD(P)H oxidase and uncoupled endothelial NO synthase (eNOS) apparently are the main sources of O(2)(*-) in the endothelial cells. It seems that O(2)(*-) generated by NAD(P)H oxidase may trigger eNOS uncoupling and contribute to the endothelial balance between NO and O(2)(*-). That is maintained at diverse levels.     

Nitric oxide mediates tightening of the endothelial barrier by ascorbic acid

Vitamin C, or ascorbic acid, decreases paracellular endothelial permeability in a process that requires rearrangement of the actin cytoskeleton. To define the proximal mechanism of this effect, we tested whether it might involve enhanced generation and/or sparing of nitric oxide (NO) by the vitamin. EA.hy926 endothelial cells cultured on semi-porous filter supports showed decreased endothelial barrier permeability to radiolabeled inulin in response to exogenous NO provided by the NO donor spermine NONOATE, as well as to activation of the downstream NO pathway by 8-bromo-cyclic GMP, a cell-penetrant cyclic GMP analog. Inhibition of endothelial nitric oxide synthase (eNOS) with N_ω-nitro-l-arginine methyl ester increased endothelial permeability, indicating a role constitutive NO generation by eNOS in maintaining the permeability barrier. Inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one also increased endothelial permeability and blocked barrier tightening by spermine NONOATE. Loading cells with what are likely physiologic concentrations of ascorbate decreased endothelial permeability. This effect was blocked by inhibition of either eNOS or guanylate cyclase, suggesting that it involved generation of NO by eNOS and subsequent NO-dependent activation of guanylate cyclase. These results show that endothelial permeability barrier function depends on constitutive generation of NO and that ascorbate-dependent tightening of this barrier involves maintaining NO through the eNOS/guanylate cyclase pathway.     

Inhibition of heat shock protein 90 (hsp90) in proliferating endothelial cells uncouples endothelial nitric oxide synthase activity

Dual increases in nitric oxide ((*)NO) and superoxide anion (O(2)(*-)) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of (*)NO generation to offset the increase in O(2)(*-) that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase (*)NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of (*)NO and O(2)(*-). Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 microM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated (*)NO production and increased L(omega)-nitroargininemethylester (L-NAME)-inhibitable O(2)(*-) generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O(2)(*-) generation, confirming that during proliferation eNOS generates (*)NO. Our findings demonstrate that hsp90 plays an important role in maintaining (*)NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit (*)NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.     

Ischemia/Reperfusion-Induced MKP-3 Impairs Endothelial NO Formation via Inactivation of ERK1/2 Pathway

Mitogen-activated protein kinase phosphatases (MKPs) are a family of dual-specificity phosphatases. Endothelial cells express multiple MKP family members, such as MKP-3. However, the effects of MKP-3 on endothelial biological processes have not yet been fully elucidated. Here, we address the association between MKP-3 and endothelial Nitric oxide (NO) formation under ischemia/reperfusion (IS/RP) condition. Human umbilical vein endothelial cells (HUVECs) were subjected to IS/RP treatment. The MKP-3 expression and NO formation were examined. IS/RP induced endothelial MKP-3 expression and inhibited eNOS expression and NO formation, accompanied by an increase of endothelial apoptosis. The siRNA experiments showed that MKP-3 was an important mediator in impairing eNOS expression and NO production in endothelial cells. Transfection of HUVECs with constitutively active ERK plasmids suggested that the above mentioned effect of MKP-3 was via inactivation of ERK1/2 pathway. Furthermore, impairment of eNOS expression was restored by treatment of histone deacetylase (HDAC) inhibitor and related to histone deacetylation and recruitment of HDAC1 to the eNOS promoter. Finally, Salvianolic acid A (SalA) markedly attenuated induction of MKP-3 and inhibition of eNOS expression and NO formation under endothelial IS/RP condition. Overall, these results for the first time demonstrated that IS/RP inhibited eNOS expression by inactivation of ERK1/2 and recruitment of HDAC1 to the gene promoter, leading to decreased NO formation through a MKP-3-dependent mechanism in endothelial cells, and SalA has therapeutic significance in protecting endothelial cells from impaired NO formation in response to IS/RP.     

Grape seed extract enhances eNOS expression and NO production through regulating calcium‐mediated AKT phosphorylation in H2O2‐treated endothelium

GSE (grape seed extract) has been shown to exhibit protective effects against cardiovascular events and atherosclerosis, although the underlying molecular mechanisms of action are unknown. Herein, we assessed the ability of GSE to enhance eNOS (endothelial nitric oxide synthase) expression and NO (nitric oxide) production in H₂O₂ (hydrogen peroxide)‐treated HUVECs (human umbilical vein endothelial cells). GSE enhanced eNOS expression and NO release in H₂O₂‐treated cells in a dose‐dependent manner. GSE inhibited intracellular ROS (reactive oxygen species) and reduced intracellular calcium in a dose‐dependent manner in H₂O₂‐treated cells, as shown by confocal microscopy. ROS was inhibited in cells pretreated with 5.0 μM GSE, 2.0 μM TG (thapsigargin) and 20.0 μM 2‐APB (2‐aminoethoxydiphenyl borate) instead of 0.25 μM extracellular calcium. In addition, GSE enhanced eNOS expression and reduced ROS production via increasing p‐AKT (AKT phosphorylation) with high extracellular calcium (13 mM). In conclusion, GSE protected against endothelial injury by up‐regulation of eNOS and NO expression via inhibiting InsP₃Rs (inositol 1,4,5‐trisphosphate receptors)‐mediated intracellular excessive calcium release and by activating p‐AKT in endothelial cells.     

Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells

Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled.     

Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models     

Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.