Insulin sensitivity is preserved despite disrupted endothelial function

It is well established that endothelial dysfunction and insulin resistance go hand in hand. However, it is unclear whether endothelial dysfunction per se is sufficient to impair insulin-mediated glucose uptake. We have previously reported that 4 wk of administration of the human immunodeficiency virus (HIV)-1 protease inhibitor indinavir to HIV-negative subjects induces endothelial dysfunction. Hence, we hypothesized that indinavir-induced endothelial dysfunction was associated with impaired insulin-mediated glucose disposal. We measured insulin-mediated glucose disposal at the level of the whole body, skeletal muscle, and vasculature by performing hyperinsulinemic euglycemic clamp, and vascular function studies, in a separate group of HIV-negative healthy nonobese subjects (n = 13) before and after 4 wk of daily oral indinavir. Four weeks of indinavir resulted in a 113 +/- 29% (P < 0.01) reduction of endothelium-dependent vasodilation, consistent with our earlier findings. In addition, there was a significant impairment of insulin-mediated vasodilation (101 +/- 14% before indinavir vs. 35 +/- 15% after indinavir; P < 0.05). However, there was no significant change in insulin-mediated glucose disposal at the level of the whole body (8.9 +/- 0.5 before indinavir vs. 8.5 +/- 0.6 mgxkg(-1)xmin(-1) after indinavir; P = 0.4), or skeletal muscle. Furthermore, in a separate group of four HIV-negative healthy nonobese subjects, we found that 4 wk of indinavir has no sustained effect on insulin-stimulated glucose uptake in adipose tissue. Thus our findings indicate that 1) endothelial dysfunction alone is insufficient to impair insulin-mediated glucose disposal, and 2) indinavir-induced endothelial dysfunction is likely due to a direct effect of the drug on the endothelium and is not coupled to the induction of insulin resistance.     

Increased expression of iNOS is associated with endothelial dysfunction and impaired pressor responsiveness in streptozotocin-induced diabetes

Studies in streptozotocin (STZ)-induced diabetic rats have demonstrated cardiovascular abnormalities such as depressed mean arterial blood pressure (MABP) and heart rate (HR), endothelial dysfunction, and attenuated pressor responses to vasoactive agents. We investigated whether these abnormalities are due to diabetes-associated activation of inducible nitric oxide synthase (iNOS). In addition, the effect of the duration of diabetes on these abnormalities was also evaluated. Diabetes was induced by administration of 60 mg/kg STZ via the tail vein. One, 3, 9, or 12 wk after STZ injection, MABP, HR, and endothelial function were measured in conscious unrestrained rats. Pressor response curves to bolus doses of methoxamine (MTX) and angiotensin II (ANG II) were constructed in the presence of N-[3(aminomethyl)benzyl]-acetamidine, dihydrochloride (1400W), a specific inhibitor of iNOS. Depressed MABP and HR and impairment of endothelial function were observed as early as 3 wk after induction of diabetes. Acute inhibition of iNOS with 1400W (3 mg/kg i.v.) restored the attenuated pressor responses to both MTX and ANG II without affecting the basal MABP and HR. Immunohistochemical and Western analysis blot studies in cardiovascular tissues revealed decreased expression of endothelial nitric oxide synthase (eNOS) concomitant with increased expression of iNOS and nitrotyrosine with the progression of diabetes. Our findings suggest that induction of iNOS in cardiovascular tissues is dependent on the duration of diabetes and contributes significantly to the depressed pressor responses to vasoactive agents and potentially to endothelial dysfunction.     

Coronary endothelial dysfunction is not rapidly reversible with ascorbic acid

In humans with cardiovascular risk factors, increased vascular production of superoxide anion may contribute to endothelial dysfunction by its reacting with nitric oxide and reducing its biological activity. High concentrations of ascorbic acid scavenge superoxide anion and restore normal endothelium-dependent vasodilation in humans with cardiovascular risk factors. To investigate the contribution of increased superoxide anion to endothelial dysfunction in atherosclerotic coronary arteries, we examined the effect of sequential infusions of ascorbic acid (final concentration 0.1, 1.0, and 10 mmol/L) or placebo on coronary endothelial function in 26 subjects referred for cardiac catheterization to evaluate coronary artery disease. Coronary vasomotor function was evaluated using intracoronary agonist infusion, quantitative angiography, and intracoronary Doppler measurements. At baseline, endothelium-dependent vasodilation of epicardial arteries and coronary microvessels was impaired to an equivalent extent in the ascorbic acid and placebo groups. Sequential ascorbic acid infusions had no effect on the acetylcholine-induced change in coronary artery diameter (-11+/-8, -12+/-10, and -9+/-9%) compared with the effect of placebo (-14+/-13, -16+/-10, and -13+/-9%) infusions (p=0.98). Similarly, the changes in coronary blood flow during acetylcholine infusions were equivalent during ascorbic acid (51+/-44, 67+/-66, and 62+/-52%) and placebo (61+/-104, 55+/-93, and 50+/-69%) infusions (p=0.63). Ascorbic acid also had no effect on the dilator response to intracoronary nitroglycerin (p=0.19). These data argue against an important role for superoxide-mediated "inactivation" of nitric oxide or another rapidly reversible form of oxidative stress as a mechanism of coronary endothelial dysfunction in patients with coronary atherosclerosis.     

Chronic administration of NMU into the paraventricular nucleus stimulates the HPA axis but does not influence food intake or body weight

C-reactive protein (CRP), a predictor of future cardiovascular diseases, has been reported to damage the vascular wall by inducing endothelial dysfunction and inflammation. This proatherogenic CRP was speculated to have a role in attenuating angiogenic functions of human endothelial progenitor cells (EPCs), possibly impairing vascular regeneration and increasing cardiovascular vulnerability to ischemic injury. Herein, we investigated the direct effect of CRP on angiogenic activity and gene expression in human EPCs. Incubation of EPCs with human recombinant CRP significantly inhibited EPC migration in response to vascular endothelial growth factor, possibly by decreasing the expression of endothelial nitric oxide synthase and subsequent nitric oxide production. In addition, CRP-treated EPCs showed the reduced adhesiveness onto an endothelial cell monolayer. When assayed for the gene expression of arteriogenic chemo-cytokines, CRP substantially decreased their expression levels in EPC, in part due to the upregulation of suppressors of cytokine signaling proteins. These results suggest that CRP directly attenuates the angiogenic and possibly arteriogenic functions of EPCs. This CRP-induced EPC dysfunction may impair the vascular regenerative capacity of EPCs, thereby leading to increased risk of cardiovascular diseases.     

Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity

Plasma adiponectin level is significantly reduced in patients with metabolic syndrome, and vascular dysfunction is an important pathological event in these patients. However, whether adiponectin may protect endothelial cells and attenuate endothelial dysfunction caused by metabolic disorders remains largely unknown. Adult rats were fed with a regular or a high-fat diet for 14 wk. The aorta was isolated, and vascular segments were incubated with vehicle or the globular domain of adiponectin (gAd; 2 mug/ml) for 4 h. The effect of gAd on endothelial function, nitric oxide (NO) and superoxide production, nitrotyrosine formation, gp91(phox) expression, and endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) activity/expression was determined. Severe endothelial dysfunction (maximal vasorelaxation in response to ACh: 70.3 +/- 3.3 vs. 95.2 +/- 2.5% in control, P < 0.01) was observed in hyperlipidemic aortic segments, and treatment with gAd significantly improved endothelial function (P < 0.01). Paradoxically, total NO production was significantly increased in hyperlipidemic vessels, and treatment with gAd slightly reduced, rather than increased, total NO production in these vessels. Treatment with gAd reduced (-78%, P < 0.01) superoxide production and peroxynitrite formation in hyperlipidemic vascular segments. Moreover, a moderate attenuation (-30%, P < 0.05) in gp91(phox) and iNOS overexpression in hyperlipidemic vessels was observed after gAd incubation. Treatment with gAd had no effect on eNOS expression but significantly increased eNOS phosphorylation (P < 0.01). Most noticeably, treatment with gAd significantly enhanced eNOS (+83%) but reduced iNOS (-70%, P < 0.01) activity in hyperlipidemic vessels. Collectively, these results demonstrated that adiponectin protects the endothelium against hyperlipidemic injury by multiple mechanisms, including promoting eNOS activity, inhibiting iNOS activity, preserving bioactive NO, and attenuating oxidative/nitrative stress.     

Dimethylarginine dimethylaminohydrolase and endothelial dysfunction in failing hearts

Congestive heart failure (CHF) is associated with impaired endothelium-dependent nitric oxide (NO)-mediated vasodilation (endothelial dysfunction). We hypothesized that coronary endothelial dysfunction in CHF may be due in part to decreased dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades endogenous inhibitors of NO synthase (NOS), including asymmetric dimethylarginine. Coronary blood flow and the endothelium-dependent vasodilator response to acetylcholine were studied in dogs in which CHF was produced by rapid ventricular pacing for 4 wk. Coronary flow and myocardial O2 consumption at rest and during treadmill exercise were decreased after development of CHF, and the vasodilator response to intracoronary acetylcholine (75 microg/min) was decreased by 39 +/- 5%. DDAH activity and DDAH isoform 2 (DDAH-2) protein content were decreased by 53 +/- 13% and 58 +/- 14%, respectively, in hearts with CHF, whereas endothelial NOS and DDAH isoform 1 (DDAH-1) were increased. Caveolin-1 and protein arginine N-methyltransferase 1, the enzyme that produces asymmetric dimethylarginine, were unchanged. Immunohistochemical staining showed DDAH-1 strongly expressed in coronary endothelium and smooth muscle and in the sarcolemma of cardiac myocytes. In cultured human endothelial cells, DDAH-1 was uniformly distributed in the cytosol and nucleus, whereas DDAH-2 was found only in the cytosol. Decreased DDAH activity and DDAH-2 protein expression may cause accumulation of endogenous inhibitors of endothelial NOS, thereby contributing to endothelial dysfunction in the failing heart.     

Arginase inhibition restores arteriolar endothelial function in Dahl rats with salt-induced hypertension

Vascular tissues express arginase that metabolizes L-arginine to L-ornithine and urea and thus reduces substrate availability for nitric oxide formation. Dahl salt-sensitive (Dahl-S) rats with salt-induced hypertension show endothelial dysfunction, including decreased vascular nitric oxide formation. This study tests the hypothesis that increased vascular arginase activity contributes to endothelial dysfunction in hypertensive Dahl-S rats. Male Dahl-S rats (5-6 wk) were placed on high (8%) or low (0.3%) NaCl diets for 4 wk. With respect to the low-salt group, mean arterial blood pressure was increased in the high-salt animals. Immunohistochemical stainings for arginase I and II were enhanced in arterioles isolated from high-salt Dahl-S rats. Experiments used isolated Krebs buffer-superfused first-order gracilis muscle arterioles with constant pressure (80 mmHg) and no luminal flow or constant midpoint but altered endpoint pressures to establish graded levels of luminal flow (0-50 microl/min). In high-salt arterioles, responses to an endothelium-dependent vasodilator acetylcholine (1 nmol/l to 3 micromol/l) and flow-induced dilation were decreased. Acute in vitro treatment with an inhibitor of arginase, 100 micromol/l (S)-(2-boronoethyl)-L-cystine, or the nitric oxide precursor, 1 mmol/l L-arginine, similarly enhanced acetylcholine and flow-induced maximal dilations and abolished the differences between high- and low-salt arterioles. These data show that arteriolar arginase expression is increased and that endothelium-dependent vasodilation is decreased in high-salt Dahl-S rats. Acute pretreatment with an arginase inhibitor or with L-arginine restores endothelium-dependent vasodilation and abolishes the differences between high- and low-salt groups. These results suggest that enhanced vascular arginase activity contributes to endothelial dysfunction in Dahl-S rats with salt-induced hypertension and identifies arginase as a potential therapeutic target to prevent endothelial dysfunction.     

Ginsenosides block HIV protease inhibitor ritonavir-induced vascular dysfunction of porcine coronary arteries

Human immunodeficiency virus (HIV) protease inhibitor ritonavir (RTV) may induce vascular dysfunction through oxidative stress. Ginsenosides have been shown to have potential benefits on the cardiovascular system through diverse mechanisms, including antioxidative property. The objective of this study was to determine whether ginsenosides could prevent coronary arteries from RTV-induced dysfunction. Porcine coronary artery rings were incubated with RTV and ginsenosides Rb1, Rc, and Re for 24 h. Vasomotor function was recorded by a myograph tension system. In response to the thromboxane A(2) analog U-46619, the contraction of the vessel rings was significantly reduced. When cocultured with Rb1, Rc, and Re, the contractility significantly increased. In response to bradykinin at 10(-5) M, the endothelium-dependent relaxation of vessel rings was significantly reduced by 59% for RTV compared with controls (P < 0.05). When cocultured with Rb1, Rc, and Re, the relaxation significantly increased 100%, 90%, and 134%, respectively, compared with the RTV-alone groups (P > 0.05). In response to sodium nitroprusside, RTV significantly reduced vasorelaxation. In addition, the endothelial nitric oxide synthase (eNOS) mRNA levels were significantly reduced by 78% for RTV group (P < 0.05) by real-time PCR analysis. The eNOS protein levels measured by Western blot analysis and nitrite concentrations measured by Griess assay were also decreased, whereas O(2)(-) production by lucigenin-enhanced chemiluminescence was significantly increased in the RTV-treated group. These effects of RTV were effectively blocked by ginsenosides. Thus HIV protease inhibitor RTV significantly impaired the vasomotor function of porcine coronary arteries. This effect may be mediated by the downregulation of eNOS and overproduction of O(2)(-). These results suggest that ginsenosides can effectively block RTV-induced vascular dysfunction.     

Effects of D-4F on vasodilation, oxidative stress, angiostatin, myocardial inflammation, and angiogenic potential in tight-skin mice

Systemic sclerosis (scleroderma, SSc) is an autoimmune, connective tissue disorder that is characterized by impaired vascular function, increased oxidative stress, inflammation of internal organs, and impaired angiogenesis. Tight skin mice (Tsk(-/+)) have a defect in fibrillin-1, resulting in replication of many of the myocardial and vascular features seen in humans with SSc. D-4F is an apolipoprotein A-I (apoA-I) mimetic that improves vascular function in diverse diseases such as hypercholesterolemia, influenza, and sickle cell disease. Tsk(-/+) mice were treated with either phosphate-buffered saline (PBS) or D-4F (1 mg.kg(-1).day(-1) for 6-8 wk). Acetylcholine and flow-induced vasodilation were examined in facialis arteries. Proinflammatory HDL (p-HDL) in murine and human plasma samples was determined by the cell-free assay. Angiostatin levels in murine and human plasma samples were determined by Western blot analysis. Hearts were examined for changes in angiostatin and autoantibodies against oxidized phosphotidylcholine (ox-PC). Angiogenic potential in thin sections of murine hearts was assessed by an in vitro vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) tube formation assay. D-4F improved endothelium-, endothelial nitric oxide synthase-dependent, and flow-mediated vasodilation in Tsk(-/+) mice. Tsk(-/+) mice had higher plasma p-HDL and angiostatin levels than C57BL/6 mice, as did SSc patients compared with healthy control subjects. Tsk(-/+) mice also had higher triglycerides than C57BL/6 mice. D-4F reduced p-HDL, angiostatin, and triglycerides in the plasma of Tsk(-/+) mice. Tsk(-/+) hearts contained notably higher levels of angiostatin and autoantibodies against ox-PC than those of control hearts. D-4F ablated angiostatin in Tsk(-/+) hearts and reduced autoantibodies against ox-PC by >50% when compared with hearts from untreated Tsk(-/+) mice. Angiogenic potential in Tsk(-/+) hearts was increased only when the Tsk(-/+) mice were treated with D-4F (1 mg.kg(-1).day(-1), 6-8 wk), and cultured sections of hearts from the D-4F-treated Tsk(-/+) mice were incubated with D-4F (10 microg/ml, 5-7 days). Failure to treat the thin sections of hearts and Tsk(-/+) mice with D-4F resulted in loss of VEGF-induced EC tube formation. D-4F improves vascular function, decreases myocardial inflammation, and restores angiogenic potential in the hearts of Tsk(-/+) mice. As SSc patients have increased plasma p-HDL and angiostatin levels similar to the Tsk(-/+) mice, D-4F may be effective at treating vascular complications in patients with SSc.     

Mechanisms of lead-induced hypertension and cardiovascular disease

Lead is a ubiquitous environmental toxin that is capable of causing numerous acute and chronic illnesses. Population studies have demonstrated a link between lead exposure and subsequent development of hypertension (HTN) and cardiovascular disease. In vivo and in vitro studies have shown that chronic lead exposure causes HTN and cardiovascular disease by promoting oxidative stress, limiting nitric oxide availability, impairing nitric oxide signaling, augmenting adrenergic activity, increasing endothelin production, altering the renin-angiotensin system, raising vasoconstrictor prostaglandins, lowering vasodilator prostaglandins, promoting inflammation, disturbing vascular smooth muscle Ca(2+) signaling, diminishing endothelium-dependent vasorelaxation, and modifying the vascular response to vasoactive agonists. Moreover, lead has been shown to cause endothelial injury, impede endothelial repair, inhibit angiogenesis, reduce endothelial cell growth, suppress proteoglycan production, stimulate vascular smooth muscle cell proliferation and phenotypic transformation, reduce tissue plasminogen activator, and raise plasminogen activator inhibitor-1 production. Via these and other actions, lead exposure causes HTN and promotes arteriosclerosis, atherosclerosis, thrombosis, and cardiovascular disease. In conclusion, studies performed in experimental animals, isolated tissues, and cultured cells have provided compelling evidence that chronic exposure to low levels of lead can cause HTN, endothelial injury/dysfunction, arteriosclerosis, and cardiovascular disease. More importantly, these studies have elucidated the cellular and molecular mechanisms of lead's action on cardiovascular/renal systems, a task that is impossible to accomplish using clinical and epidemiological investigations alone.     

Role of oxidative stress in multiparity-induced endothelial dysfunction

Multiparity is associated with increased risk of cardiovascular disease. We tested whether multiparity induces oxidative stress in rat vascular tissue. Coronary arteries and thoracic aorta were isolated from multiparous and age-matched virgin rats. Relaxation to ACh and sodium nitroprusside (SNP) was measured by wire myography. We also tested the effect of the superoxide dismutase mimetic MnTE2PyP (30 microM), the NADPH oxidase inhibitor apocynin (10 microM), and the peroxynitrite scavenger FeTPPs (10 microM) on ACh-mediated relaxation in coronary arteries. Vascular superoxide anion was measured using the luminol derivative L-012 and nitric oxide (NO) generation by the Griess reaction. Multiparity reduced maximal response and sensitivity to ACh in coronary arteries [maximal relaxation (E(max)): multiparous 49+/-3% vs. virgins 95%+/-3%; EC(50): multiparous 135+/-1 nM vs. virgins 60+/-1 nM], and in aortic rings (E(max): multiparous 38+/-3% vs. virgins 79+/-4%; EC(50): multiparous 160+/-2 nM vs. virgins 90+/-3 nM). Coronary arteries from the two groups relaxed similarly to SNP. Superoxide anions formation was significantly higher in both coronary arteries (2.8-fold increase) and aorta (4.1-fold increase) from multiparous rats compared with virgins. In multiparous rats, incubation with MnTE2PyP, apocynin, and FeTPPs improved maximal relaxation to ACh (MnTE2PyP: 74+/-5%; vehicle: 41+/-5%; apocynin: 73+/-3% vs. vehicle: 41+/-3%; FeTPPs: 72+/-3% vs. vehicle: 46+/-3%) and increased sensitivity (EC(50): MnTE2PyP: 61+/-0.5 nM vs. vehicle: 91+/-1 nM; apocynin: 45+/-3 nM vs. vehicle: 91+/-6 nM; FeTPP: 131 +/- 2 nM vs. vehicle: 185+/-1 nM). Multiparity also reduced total nitrate/nitrite levels (multiparous: 2.5+/-2 micromol/mg protein vs. virgins: 7+/-1 micromol/mg protein) and endothelial nitric oxide synthase protein levels (multiparous: 0.53+/-0.1 protein/actin vs. virgins: 1.0+/-0.14 protein/actin). These data suggest that multiparity induces endothelial dysfunction through decreased NO bioavailability and increased reactive oxygen species formation.     

Pro-angiogenic activity of Urotensin-II on different human vascular endothelial cell populations

Urotensin-II (U-II), along its receptor UT, is widely expressed in the cardiovascular system, where it exerts regulatory actions under both physiological and pathological conditions. In the present study, human vascular endothelial cells (EC) from one arterious and three venous vascular beds were used to investigate in vitro their heterogeneity in terms of expression of U-II and UT and of angiogenic response to the peptide. Real-time PCR and immunocytochemistry demonstrated the expression of UT, as mRNA and protein, in all the EC populations investigated. U-II, on the contrary, was detectable only in EC from aorta and umbilical vein. U-II did not affect the proliferation rate of adult human EC, but induced a moderate proliferative effect on EC from human umbilical vein. When tested in the Matrigel assay, however, all EC exhibited a strong angiogenic response to the peptide, comparable to that of fibroblast growth factor-2 (FGF-2) and it was not associated to an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. The angiogenic effect of U-II was abolished by the UT antagonist palosuran. Overall, these data suggest that U-II, in addition to the well known role in the regulation of cardiovascular function, also exert a specific angiogenic activity.     

Effect of age and gender on the progression of adult vascular dysfunction in a mouse model of fetal programming lacking endothelial nitric oxide synthase

The objective of this study was to investigate vascular function at different ages in a transgenic murine model of fetal vascular programming using a model of uteroplacental insufficiency induced by lack of endothelial nitric oxide synthase. Homozygous NOS3 knockout (KO) and wild-type (WT) mice were cross bred to produce WT, KO, and heterozygous that developed in WT (KOP) or KO (KOM) mothers. Male/female offspring from the four groups were killed at 7, 14, and 21 wk of age (n = 5-10/group), and carotid arteries were used for in vitro vascular studies. Responses to phenylephrine (PE), with/without N(G)-nitro-L-arginine methyl ester (L-NAME), angiotensin (ANG), acetylcholine (ACh), sodium nitroprusside, and isoproterenol (ISO) were studied. At 7 wk, only KO offspring showed higher contractile response to PE, whereas, at 14 and 21 wk, both KO and KOM had a higher response. Incubation with L-NAME abolished these differences. ANG contraction was higher in male KO in all age groups and in 21-wk-old females. Relaxation to ACh and ISO was absent in KO, and significantly decreased in KOM offspring in all age groups compared with KOP and WT, independent of gender. Sodium nitroprusside was not different between groups. The effect of the altered intrauterine environment on the development of abnormal vascular function was limited at 7 wk of age and most evident at 14 wk; further deterioration was limited to ANG-mediated vascular contractility in KO offspring. Our findings provide some hope that at least the first seven postnatal weeks may be an appropriate therapeutic window to prevent cardiovascular disease later in life.     

Type 2 Diabetes: Endothelial dysfunction and Exercise

[Purpose]

Vascular endothelial dysfunction is an early marker of atherosclerosis characterized by decreased nitric oxide bioavailability in the vascular endothelium and smooth muscle cells. Recently, some animal models and in vitro trials demonstrated that excessive superoxide production from mitochondria within vascular endothelial cells played a role in the pathogenesis of atherosclerosis in type 2 diabetes. This review provides a systematic assessment of the effectiveness of exercise to identify effective approaches to recognize diabetes risk and prevent progression to heart disease.

[Methods]

A systematic literature search was conducted to retrieve articles from 1979 to 2013 using the following databases: the MEDLINE, PubMed. Articles had to describe an intervention that physical activity and exercise to identify effective approaches to heart and vascular endothelium.

[Results]

Currently, physical activity and exercise guidelines aimed to improve cardiovascular health in patients with type 2 diabetes are nonspecific. Benefit of aerobic exercise training on vascular endothelial function in type 2 diabetic patients is still controversial.

[Conclusion]

it is necessary to demonstrate the mechanism of endothelial dysfunction from live human tissues so that we can provide more specific exercise training regimens to enhance cardiovascular health in type 2 diabetic patients.     

Glycogen synthase kinase-3β is involved in C-reactive protein-induced endothelial cell activation

C-reactive protein (CRP) is a significant contributor to atherosclerosis and a powerful predictor of cardiovascular risk. The role of CRP in endothelial cell (EC) activation has been extensively investigated, but the underlying mechanisms have not been fully elucidated. The effect of glycogen synthase kinase-3β (GSK-3β) on CRP-induced EC activation was evaluated in this study. We observed that CRP decreased endothelial nitric oxide synthase (eNOS) activity during EC activation. CRP also activated GSK-3β by dephosphorylating its Ser9 level and reducing β-catenin protein expression in a time-dependent manner. We also found that the GSK-3β inhibitors TDZD-8 and SB415286 partially restored eNOS activity and suppressed the release of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 from ECs. These data provide new evidence for the involvement of GSK-3β in EC activation.     

Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans

A reduced nitric oxide availability is a hallmark of endothelial dysfunction occurring early in atherosclerosis. Recently, we have shown that plasma nitrite mirrors acute changes in endothelial nitric oxide synthase activity in various mammals, including humans. Here, we examined the hypothesis that plasma nitrite levels are reduced in humans with endothelial dysfunction and the decrease is correlated with increasing numbers of cardiovascular risk factors (RF). Plasma nitrite concentrations were quantified by flow-injection analysis. The coefficient of variation for repeated measurements of plasma nitrite was <8%, and heart rate and blood pressure at the time of blood sampling had no significant effect on nitrite values measured (n=10). Baseline levels of plasma nitrite followed a normal distribution in each group studied and decreased progressively with increasing numbers of cardiovascular risk factors (n=351, p<0.001): 351+/-13 (0 RF), 261+/-10 (1 RF), 253+/-11 (2 RF), 222+/-18 (3 RF), and 171+/-29 nmol/L (4 RF). Intima media thickness (IMT) and flow-mediated dilation (FMD) were determined via ultrasound. Plasma nitrite and FMD levels were lower, whereas IMT was greater in individuals with endothelial dysfunction (n=12) compared to healthy volunteers (n=12). Nitrite correlated significantly with FMD (r=0.56, p<0.001) and inversely with IMT (r= -0.49, p<0.01). Plasma nitrite levels are reliably measurable in humans, indicate endothelial dysfunction, and correlate with cardiovascular risk factors. Future studies are necessary to identify the prognostic relevance of plasma nitrite determination in patients suffering from cardiovascular disease.     

Fish oil and vascular endothelial protection: bench to bedside

Fish oil is recommended for the management of hypertriglyceridemia and to prevent secondary cardiovascular disorders. Fish oil is a major source of ω-3-polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Clinical studies suggest that fish oil not only prevents the incidence of detrimental cardiovascular events, but also lowers the cardiovascular mortality rate. In addition to a classic lipid-lowering action, ω-3-PUFAs in fish oil could regulate blood pressure and enhance vascular integrity and compliance. Additionally, ω-3-PUFAs have the ability to protect vascular endothelial cells by decreasing oxidative stress, halting atherosclerotic events, and preventing vascular inflammatory and adhesion cascades. Intriguingly, recent studies have demonstrated that ω-3-PUFAs improve the function of vascular endothelium by enhancing the generation and bioavailability of endothelium-derived relaxing factor (nitric oxide) through upregulation and activation of endothelial nitric oxide synthase (eNOS). This certainly opens up a new area of research identifying potential mechanisms influencing fish oil-mediated functional regulatory action on vascular endothelium. We address in this review the potential of fish oil to prevent vascular endothelial dysfunction and associated cardiovascular disorders. Moreover, the mechanisms pertaining to fish oil-mediated eNOS activation and nitric oxide generation in improving endothelial function are delineated. We finally suggest the importance of further studies to determine the dose adjustment of fish oil with an optimal ratio of EPA and DHA for achieving consistent cardiovascular protection.     

Evidence against oxidative stress as mechanism of endothelial dysfunction in methionine loading model

Endothelial dysfunction reflects reduced nitric oxide (NO) bioavailability due to either reduced production, inactivation of NO, or reduced smooth muscle responsiveness. Oral methionine loading causes acute endothelial dysfunction in healthy subjects and provides a model in which to study mechanisms. Endothelial function was assessed using flow-mediated dilatation (FMD) of the brachial artery in humans. Three markers of oxidative stress were measured ex vivo in venous blood. NO responsiveness was assessed in vascular smooth muscle and platelets. Oral methionine loading induced endothelial dysfunction (FMD decreased from 2.8 +/- 0.8 to 0.3 +/- 0.3% with methionine and from 2.8 +/- 0.8 to 1.3 +/- 0.3% with placebo; P < 0.05). No significant changes in measures of plasma oxidative stress or in vascular or platelet sensitivity to submaximal doses of NO donors were detected. These data suggest that oxidative stress is not the mechanism of endothelial dysfunction after oral methionine loading. Furthermore, the preservation of vascular and platelet NO sensitivity makes a signal transduction abnormality unlikely.     

Bradykinin induces superoxide anion release from human endothelial cells

The time-dependent release of superoxide anion (O2-) from bradykinin (Bk)-stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome C employing a novel application of microspectrophotometry. In the absence of Bk, O2- release by EC was not detectable. EC exposure to Bk (10(-6) to 10(-5) M) resulted in a rapid release of O2-. The release of O2- occurred within 5 minutes of exposure. O2- release was partially inhibited by indomethacin (63 +/- 6%), thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC O2- production. EC O2- release may be an important component in the pathophysiologic actions of Bk on vascular function.     

Restoration of endothelin-1-induced impairment in endothelium-dependent relaxation by interleukin-10 in murine aortic rings

Endothelin-1 (ET-1) is implicated in the development of endothelial dysfunction through the generation of reactive oxygen species by NADPH oxidase activation. Interleukin-10 (IL-10) is an antiinflammatory cytokine that stimulates nitric oxide production, decreases superoxide production, and restores endothelial integrity after vascular injury. In this study, we tested whether IL-10 attenuates ET-1-induced endothelial dysfunction by improving acetylcholine (ACh)-induced relaxation of cultured murine aortic rings. Aortic rings (2 mm long) of C57BL/6 mice were incubated in 2 mL DMEM containing 120 U/mL penicillin and 120 mug/mL streptomycin in the presence of one of 4 treatments: vehicle (deionized water), ET-1 (100 nmol/L), recombinant mouse IL-10 (300 ng/mL), or a combination of both ET-1 and IL-10. After incubation at 37 degrees C for either 1 or 6 h (short-term exposure) or 22 h (overnight exposure), rings were mounted in a wire myograph and stretched to a passive force of 5 mN. Endothelium-dependent vasorelaxation was assessed by constructing cumulative concentration-response curves to ACh (0.001-10 mumol/L) during 10 mumol/L phenylephrine (PE)-induced contraction. Short-term exposure of ET-1 did not result in an impairment of ACh-induced relaxation. Overnight exposure of aortic rings to ET-1 resulted in a statistically significant endothelial dysfunction characterized by a reduced maximal relaxation response to ACh compared with that of untreated rings (Emax 57% +/- 3% versus 82% +/- 4%). IL-10 treatment restored ACh-induced relaxation (Emax 77% +/- 3%). Western blotting showed decreased eNOS expression in response to ET-1, whereas vessels treated with a combination of ET-1 and IL-10 showed increased expression of eNOS. Immunohistochemical analysis showed decreased eNOS expression in ET-1-treated vessels compared with those treated with both ET-1 and IL-10. We conclude that, in murine aorta, the antiinflammatory cytokine IL-10 prevents impairment in endothelium-dependent relaxation induced in response to long-term incubation with ET-1 via normalization of eNOS expression.