Oxide Synthesis In Endothelial Cells Research Articles

Small extracellular vesicles from pregnant women with maternal supraphysiological hypercholesterolemia impair endothelial cell function in vitro

Maternal physiological hypercholesterolemia MPH, maternal total cholesterol (TC) levels at term of pregnancy ≤280 mg/dL) occurs to assure fetal development. Maternal supraphysiological hypercholesterolemia (MSPH, TC levels >280 mg/dL) is a pathological condition associated with maternal, placental, and fetal endothelial dysfunction and early neonatal atherosclerosis development. Small extracellular vesicles (sEVs) are delivered to the extracellular space by different cells, where they modulate cell functions by transporting active signaling molecules, including proteins and miRNA. AimTo determine whether sEVs from MSPH women could alter the function of endothelial cells (angiogenesis, endothelial activation and nitric oxide synthesis capacity). MethodsThis study included 24 Chilean women (12 MPH and 12 MSPH). sEVs were isolated from maternal plasma and characterized by sEV markers (CD9, Alix and HSP70), nanoparticle tracking analysis, transmission electron microscopy, and protein and cholesterol content. The endothelial cell line HMEC-1 was used to determine the uptake of labeled sEVs and the effects of sEVs on cell viability, endothelial tube formation, endothelial cell activation, and endothelial nitric oxide expression and function. ResultsIn MSPH women, the plasma concentration of sEVs was increased compared to that in MPH women. MSPH-sEVs were highly taken up by HMEC-1 cells and reduced angiogenic capacity and the expression and activity of eNOS without changing cell viability or endothelial activation compared to MPH-sEVs. ConclusionsEVs from MSPH women impair angiogenesis and nitric oxide synthesis in endothelial cells, which could contribute to MSPH-associated endothelial dysfunction.

Harnessing hyperuricemia to atherosclerosis and understanding its mechanistic dependence.

Atherosclerosis is regarded as the disease of the arterial vasculature. The main characteristics of atherosclerosis are the abnormal accumulation of lipids, increased inflammatory cells, matrix deposits, and proliferation of smooth muscle cells. Diabetes mellitus, obesity, and hyperlipidemia are the most studied risk factors of atherosclerosis. One least studied risk factor is the uric acid (UA), a high UA in circulation is interlinked with many pathological processes. Several epidemiological studies suggest elevated UA levels as an essential biomarker in the forecast of several cardiovascular diseases. Available evidence claims that UA upholds the atherosclerosis process via disturbing lipid metabolism, reducing the nitric oxide synthesis in endothelial cells, promoting the proliferation of vascular smooth muscle cells, and overwhelms inflammation. In endothelial dysfunction and coronary artery lesions, UA is considered as an independent predictor. The updated studies on the involvement of hyperuricemia in atherosclerosis prove that treatment with xanthine oxidase (XO) inhibitors not just benefits the treatment of hyperuricemia but also reduces the burden of atherosclerosis to a greater extent. In this review, we highlight how the hyperuricemia affects vascular integrity, causes atherosclerosis, and the mechanism of action of XO inhibitors on atherosclerosis.

Thiamine pyrophosphate diminishes nitric oxide synthesis in endothelial cells.

Although thiamine pyrophosphate (TPP) is considered a protective agent for endothelial cells, it is still unknown if this is associated with nitric oxide (NO) synthesis. Our aim was to evaluate the synthesis of NO in endothelial cells incubated with TPP and high glucose concentrations. Endothelial cells from the umbilical cord vein from newborns (n=20), were incubated with 5, 15 or 30mmol/L glucose, in absence or presence of 0.625mg/ml of TPP. Our results showed a significant increase in cell proliferation (> 40%; P<0.05), and cell viability (> 90%; P<0.001) after 48h in endothelial cells cultured with glucose plus TPP. Likewise, in the presence of glucose and TPP an important rise in the consumption of glucose by the endothelial cells was observed after 24h (> 7%; P<0.001) and 48h (> 10%; P<0.05). Additionally, the levels of lactate after incubation with glucose and TPP showed only slight variations after 48h (P<0.05). However, these changes were clearly different from those observed in the absence of TPP. Interestingly, we found that the changes mentioned were linked with reduced levels of nitrites both at 24h (< 171 pmol/μg protein; P<0.001), and 48h (< 250 pmol/μg protein; P<0.05), which was associated with a reduced expression of mRNA of eNOS in endothelial cells incubated with TPP and high glucose. In conclusion, the presence of TPP regulates the consumption of glucose and the synthesis of NO, which would explain its protective effect in the endothelium of diabetic patients.

A comparative analysis of the level of high-sensitivity C-reactive protein in individuals with and without hypertension

Objective The objective of this study was to compare the level of serum high-sensitivity C-reactive protein (hs-CRP) in hypertensive and normotensive individuals. Background Hs-CRP production is part of the nonspecific acute-phase response to most forms of inflammation, infection, and tissue damage. The association between hs-CRP and hypertension (HTN) could be related in part as follows: correlation between elevated CRP and arterial stiffness; association between CRP and metabolic syndrome, one of whose criteria is HTN and the possibility that CRP may directly contribute to reduced nitric oxide synthesis in endothelial cells, leading to increased vascular resistance. Patients and methods Our study comprised 80 patients, divided into two groups: the first group consisted of 55 patients with blood pressure more than140/90 mmHg recorded in at least two separate clinic-based measurements after ruling out secondary causes. The second group consisted of 25 normotensive individuals with a blood pressure of 100–120/60–80 mmHg as a control group. All individuals were subjected to full history taking, complete medical examination, 12-lead ECG, conventional echocardiography, routine laboratory investigations, and measurement of hs-CRP by CRP HS enzyme-linked immunosorbent assay. Results The study showed that the mean age of the studied group was 43.9 years; 58.8% were male individuals. Mean systolic and diastolic blood pressure was 146.4 and 91.5 mmHg, respectively. There were highly significant differences between the patient and control groups with regard to age, blood pressure, left ventricular mass, left ventricular mass index, lipid profile, and hs-CRP (higher in the patient group). The study showed also highly significant positive correlations between hs-CRP and age, blood pressure, left ventricular mass and left ventricular mass index. There was a significant positive correlation between hs-CRP and total cholesterol. Conclusion The levels of hs-CRP were elevated in hypertensive individuals, which suggests the possibility of an inflammatory pathogenesis in HTN.

Propionyl-L-carnitine induces eNOS activation and nitric oxide synthesis in endothelial cells via PI3 and Akt kinases

Propionyl-l-carnitine (PLC) is a natural short-chain derivative of l-carnitine (LC), a natural amino acid that plays an important role in fatty acid metabolism. Recent studies suggest that PLC has vascular protective effects. Because of the importance of endothelial nitric oxide synthase (eNOS) and its product, antiatherogenic molecule nitric oxide (NO), in vascular endothelial function, we sought to elucidate that if PLC would stimulate eNOS and its upstream activators Akt and phosphatidylinositol 3-kinase (PI3 Kinase) in cultured human aortic endothelial cells (HAEC). PLC caused eNOS phosphorylation at Ser-1177, and dominant negative Akt and a novel Akt-selective inhibitor MK-2206 inhibited both PLC-mediated phosphorylation and activation of the enzyme. PI3 kinase inhibition also blocked the phosphorylation and activation of eNOS by PLC. Studies with specific drug inhibitors PD173955 and PP2 showed that the non-receptor tyrosine kinase, src, is an upstream stimulator of the PI3 kinase-Akt pathway in this pathway. In addition, PLC significantly decreased intracellular ATP/ADP ratio and activate AMPK, subsequently leading to Src activation. Finally, we demonstrated that the effects of PLC to augment eNOS activity were associated with a net increase in NO release from endothelial cells. NO production following incubation with PLC was abolished in endothelial cells coincubated with L-NAME, PD173955, LY294002, MK-2206 and compound C. In conclusion, PLC, via AMPK/Src-mediated signaling that leads to activation of PI3 kinase and Akt, stimulates eNOS, leading to increased production of NO.

L‐Leucine Inhibits Nitric Oxide Synthesis in Endothelial Cells

Concentrations of L‐leucine (physiological isomer) are elevated in the plasma of obese humans as well as both diet‐induced and spontaneously obese rats in association with vascular insulin resistance. Because reduced availability of nitric oxide (NO) has been implicated to play a key role in mediating the metabolic syndrome in obese subjects, this study was conducted to test the hypothesis that elevated levels of L‐leucine may inhibit NO synthesis by endothelial cells (EC). Bovine venular EC were cultured for 48 h in Dulbecco's modified Eagle's medium containing 0.2 mM L‐arginine, 0.5 mM L‐glutamine and 0.1, 0.5 or 2 mM L‐leucine, L‐valine, L‐isoleucine, or D‐leucine. L‐Leucine dose‐dependently increased (P &lt; 0.05) glutamine:fructose aminotransferase (GFAT) activity and the synthesis of glucosamine‐6‐P (a competitive inhibitor of glucose‐6‐P dehydrogenase, an enzyme in the pentose cycle), while inhibiting NO production by EC (P &lt; 0.05). L‐Valine, L‐isoleucine, and D‐leucine had no effect on GFAT activity or NO synthesis in these cells (P &gt;; 0.05). Compared with 0.1 mM L‐leucine, 0.5 and 2 mM L‐leucine reduced pentose cycle activity and intracellular concentrations of NADPH (a cofactor for NO synthase) in a dose‐dependent manner. These results suggest that L‐leucine inhibits NO production by endothelial cells through glucosamine‐mediated reduction of NADPH generation via the pentose cycle. Supported by AHA.

Folate intake and incidence of hypertension among American young adults: a 20-y follow-up study

Laboratory studies suggest that folate intake may decrease blood pressure (BP) through increasing nitric oxide synthesis in endothelial cells and/or reducing plasma homocysteine concentrations. However, human studies, particularly longitudinal data, are limited. Our objective was to investigate whether dietary folate intake is associated with the 20-y incidence of hypertension. We prospectively followed 4400 men and women (African Americans and whites aged 18-30 y) without hypertension at baseline (1985) in the Coronary Artery Risk Development in Young Adults study 6 times, in 1987, 1990, 1992, 1995, 2000, and 2005. Diet was assessed by dietary-history questionnaire at baseline and in 1992 and 2005. Incident hypertension was defined as the first occurrence at any follow-up examination of systolic BP ≥ 140 mm Hg, diastolic BP ≥ 90 mm Hg, or use of antihypertensive medication. A total of 989 incident cases were identified during the 20-y follow-up. After adjustment for potential confounders, participants in the highest quintile of total folate intake had a significantly lower incidence of hypertension (HR: 0.48; 95% CI: 0.38, 0.62; P-trend < 0.01) than did those in the lowest quintile. The multivariable HRs for the same comparison were 0.33 (95% CI: 0.22, 0.51; P-trend < 0.01) in whites and 0.54 (95% CI: 0.40, 0.75; P-trend < 0.01) in African Americans (P-interaction = 0.047). The inverse associations were confirmed in a subset of the cohort (n = 1445) with serum folate measured at baseline and in 1992 and 2000. Higher folate intake in young adulthood was longitudinally associated with a lower incidence of hypertension later in life. This inverse association was more pronounced in whites. Additional studies are warranted to establish the causal inference.

Angiogenin Induces Nitric Oxide Synthesis in Endothelial Cells through PI-3 and Akt Kinases

Angiogenesis, the formation of new blood vessels, is a critical but complex phenomenon modulated by numerous physicochemical conditions. Nitric oxide (NO) is a very well known biological mediator involved in vascular physiology. This study focuses on relationships between the effect of angiogenin, a major angiogenic factor, and extracellular NO release. NO concentration was sensed electrochemically using a fibronectin-coated multiple microelectrode array. Angiogenin was shown to increase NO levels, thus triggering nitric oxide synthase (NOS) activity. The effect of angiogenin on NOS was demonstrated using l-NAME, a competitive NOS inhibitor. Dose-time dependence was investigated, showing a stimulation threshold in the 250 ng/mL-1 microg/mL range and a maximal NO release after 30 min of exposure to angiogenin. To elucidate the very complex reactive pathway of angiogenin, we have used various selective inhibitors to investigate the mechanism leading to NO production. Neomycin, an antibiotic blocking nuclear translocation, inhibited the angiogenin effect on NOS. This result demonstrates that angiogenin activates NOS by interacting with the cell nucleus. Similarly, NOS activity was stopped by blocking the PI-3/Akt kinase signaling transduction cascade, showing the importance of this pathway.

GTP cyclohydrolase I gene transfer reverses tetrahydrobiopterin deficiency and increases nitric oxide synthesis in endothelial cells and isolated vessels from diabetic rats.

Nitric oxide (NO) synthesis in endothelial cells is impaired in diabetes. We previously showed that impaired NO synthesis in the spontaneously diabetic BB (BBd) rat is due to decreased levels of tetrahydrobiopterin (BH4), secondary to decreased expression of GTP cyclohydrolase I (GTPCH). The aim of this study was to utilize adenoviral GTPCH gene transfer to reverse BH4 deficiency and repair the ability of endothelial cells to produce NO. GTPCH gene transfer increased BH4 levels in BBd endothelial cells from 0.17 +/- 0.02 (mean +/-SE) to 73.37 +/- 14.42 pmol/million cells and NO production from 0.77 +/- 0.07 to 18.74 +/- 5.52 nmol/24 h/million cells. To demonstrate a functional effect of increasing BH4 concentrations in tissues, we transferred GTPCH into aortic rings from BBd and Zucker diabetic fatty (ZDF) rats, models of human type I and type II diabetes, respectively. GTPCH gene transfer led to a dose-dependent increase in acetylcholine-induced vasorelaxation, preventable by inhibiting NO synthase. Maximal relaxation of virus-treated rings (10(10) virus particles/ml) to acetylcholine was significantly higher than sham-treated rings (BBd 64% vs. 37%, P<0.005; ZDF 80% vs. 44%, P<0.05). This study demonstrates that GTPCH gene transfer can reverse BH4 deficiency in both type I and type II diabetes and provides an experimental basis for using gene therapy to treat cardiovascular complications in diabetic patients.

Genomic and non-genomic effects of estrogens on endothelial cells

Estrogen receptors act via the regulation of transcriptional processes, involving nuclear translocation and binding on specific response elements, thus leading to regulation of target gene expression. However, novel non-transcriptional mechanisms of signal transduction through steroid hormone receptors have been identified. These so-called “non-genomic” effects are independent by gene transcription or protein synthesis and involve steroid-induced modulation of cytoplasmic or of cell membrane-bound regulatory proteins. Relevant biological actions of steroids have been associated with this signaling in different tissues. Ubiquitary regulatory cascades such as mitogen-activated protein kinases (MAPK), the phosphatidylinositol 3-OH kinase (PI3K) and tyrosine kinases are modulated through non-transcriptional mechanisms by steroid hormones. Furthermore, steroid hormone receptors modulation of cell membrane-associated molecules such as ion channels and G-protein-coupled receptors has been shown in diverse tissues. The vascular wall is a site where non-genomic steroid hormones actions are particularly prominent. For instance, estrogens and glucocorticoids trigger rapid vasodilatation due to rapid induction of nitric oxide synthesis in endothelial cells via the estrogen receptor-dependent activation of MAPK and PI3K, leading to relevant pathophysiological consequences, in vitro and in vivo. The growing amount of evidence collected in the last years claims that non-transcriptional signaling mechanisms play a primary role in the generation of the effects of steroids on endothelial cells, which may turn out to be of relevance for clinical purposes.

The influence of lipid-lowering treatment on nitric oxide synthesis in endothelial cells

The influence of lipid-lowering treatment on nitric oxide synthesis in endothelial cells

Role of nitric oxide and acetylcholine in neocortical hyperemia elicited by basal forebrain stimulation: evidence for an involvement of endothelial nitric oxide.

We examined the role of acetylcholine and nitric oxide in the increases in cerebrocortical blood flow elicited by stimulation of a region of the basal forebrain from which the major cholinergic projection to the cerebral cortex originates. In halothane-anesthetized rats a 3 x 3 mm area of the parietal cortex was exposed and the site was superfused with Ringer (37 degrees C; pH 7.3-7). Cortical blood flow was monitored at the site of superfusion by laser-Doppler flowmetry. The basal forebrain was stimulated electrically (100 microA; 50 Hz) and stimulated sites were histologically verified at the end of the experiment. With Ringer superfusion (n = 8), basal forebrain stimulation increased neocortical flow by 185 +/- 9% (mean +/- S.E.M.). The flow increase was attenuated (-38 +/- 6%; n = 5) by superfusion with the muscarinic cholinergic antagonist atropine (100 microM). Superfusion with atropine plus the nicotinic antagonist mecamylamine (100 microM) did not attenuate the response further (P > 0.05 from atropine alone; n = 6). Superfusion with the nitric oxide synthase inhibitor nitro-L-arginine, but not with the inactive isomer nitro-D-arginine (n = 6), attenuated the vasodilation in a dose-dependent fashion (-43 +/- 4% at 1 mM; n = 7) and reduced nitric oxide synthase catalytic activity at the site of superfusion by 95 +/- 4%. Co-application of nitro-L-arginine and atropine did not attenuate the vasodilation further (P > 0.05 from nitro-L-arginine alone; n = 6). Administration of the somewhat selective inhibitor of neuronal nitric oxide synthase 7-nitroindazole (50 mg/kg, i.p.) attenuated the increases in flow produced by topical application of N-methyl-D-aspartate (40 microM; n = 5) or by hypercapnia (n = 7), but did not affect the vasodilation produced by basal forebrain stimulation (n = 5) and by topical application of acetylcholine (10 microM; n = 5). 7-nitroindazole reduced constitutive nitric oxide synthase enzymatic activity in forebrain by 72 +/- 3% (n = 8). The data suggest that the neocortical vasodilation elicited by basal forebrain stimulation is, in part, mediated by local release of acetylcholine which, in turn, leads to increased nitric oxide synthesis in endothelial cells.

Intracellular oxidative stress induced by nitric oxide synthesis inhibition increases endothelial cell adhesion to neutrophils.

The objective of the present study was to determine whether prolonged inhibition of nitric oxide synthesis in endothelial cells increased the surface adhesion of these cells for neutrophils. Human umbilical vein endothelial cells (HUVECs) were grown to confluence in 48-well microtiter plates. Exposure of HUVECs to the nitric oxide synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) did not cause neutrophil adhesion at 1 hour but increased adhesion at 4 hours in a dose-dependent manner. The increased adhesion was prevented with L-arginine or nitric oxide donors but not an analogue of cGMP. The increased adhesion was inhibited by monoclonal antibodies directed against the beta 2-integrin CD18 and endothelial cell adhesion molecule ICAM-1. Platelet-activating factor (PAF) receptor antagonist WEB 2086 also prevented the L-NAME-induced neutrophil adhesion. Intracellular oxygen radical scavengers (dimethyl sulfoxide, butylated hydroxytoluene, and alpha, alpha'-dipyridyl), the iron chelator desferrioxamine, and the mitochondrial inhibitor azide inhibited the L-NAME-induced neutrophil adhesion, whereas extracellular oxygen radical scavengers (superoxide dismutase and catalase) had no effect. HUVECs were loaded with 2',7'-dichlorodihydrofluorescein diacetate, and oxidation to the fluorescent dichlorodihydrofluorescein (DCHF) was monitored. Fluorescence was enhanced in the L-NAME-treated HUVECs throughout the 4-hour incubation, an event inhibitable by an antioxidant and azide. The magnitude of the intracellular oxidation of DCHF was equivalent to approximately 0.8 mumol/L H2O2. These data suggest that prolonged nitric oxide synthesis inhibition in HUVECs causes an oxidant- and PAF-associated rise in adhesion on the surface of these endothelial cells for neutrophils.

Endothelin-3 Modulates Glomerular Filtration Rate in the Isolated Perfused Rat Kidney

The present study has been performed to evaluate hemodynamic and tubular effects of various endothelin-3 (ET-3) concentrations on the isolated perfused rat kidney. Using this experimental system we observed a profound reduction of renal perfusate flow at all ET-3 concentrations tested (50, 250 and 500 pmol/l), suggesting that the vasoconstrictive potency of ET-3 in the kidney is comparable to that described for endothelin-1 (ET-1). The effects on glomerular filtration rate (GFR) differed depending on the ET-3 dose applied. While 250 pmol/l ET-3 increased GFR by 30%, 500 pmol/l ET-3 markedly reduced GFR. 50 pmol/l ET-3 did not alter GFR although renal vascular resistance significantly increased. Infusion of 1 mumol/l N-nitro-L-arginine, a specific and potent inhibitor of nitric oxide synthesis in endothelial cells, abolished the GFR elevation induced by 250 pmol/l ET-3. In parallel with the changes of GFR we observed an increase in sodium reabsorption at 250 pmol/l and a decrease of this parameter at 500 pmol/l ET-3. Moreover, an ET-3 concentration (500 pmol/l), which induced a dramatic fall in tubular sodium load, led to an increase of fractional sodium excretion and to a decrease of renal oxygen consumption. We conclude that ET-3 is a potent vasoconstrictor in the isolated perfused rat kidney. Furthermore, it modulates GFR in a differentiated mode, depending on the concentration used. The GFR increase at 250 pmol/l ET-3 seems to be mediated by endothelium-derived nitric oxide. In addition to its glomerular action, ET-3 might also affect tubular sodium transport.

Nitric oxide synthesis in endothelial cells: evidence for a pathway inducible by TNF-alpha.

Nitric oxide (NO) release accounts for the biological activity of endothelium-derived relaxing factor. Given that tumor necrosis factor-alpha (TNF-alpha) has been implicated as an important mediator in septic shock, we explored whether TNF-alpha enhances L-arginine-dependent synthesis of NO and L-citrulline in endothelial cells. The release of NO was detected in a coincubation bioassay where measurement of guanosine 3',5'-cyclic monophosphate (cGMP) production in reporter monolayers, namely glomerular mesangial cells or fetal lung fibroblasts, reflected activation of soluble guanylate cyclase. Reporter monolayer cGMP content was greater in the presence of TNF-alpha-treated bovine aortic and renal artery endothelial cells than in the presence of vehicle-treated endothelial cells. TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were first evident at 8 h and maximal at 16-24 h. In addition, TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were abrogated by hemoglobin and methylene blue, blunted by N omega-nitro-L-arginine and augmented by superoxide dismutase and the calcium agonist bradykinin. These observations suggested that TNF-alpha enhanced release of NO. Furthermore, the formation of L-[14C]citrulline from L-[14C]arginine, as determined by quantitative cation-exchange chromatography and thin-layer chromatography, was enhanced by TNF-alpha in a time- and concentration-dependent manner. Thus it is evident that endothelial cells release NO for a prolonged period in response to TNF-alpha and transiently when stimulated with calcium agonists. The prolonged release of NO from TNF-alpha-stimulated endothelial cells may be implicated in the pathogenesis of septic shock.