cyclic-gmp and Hypercholesterolemia

Soluble guanylyl cyclase (sGC) is a key enzyme of the NO-cGMP pathway which is believed to mediate vasoprotective actions. In cardiovascular diseases such as hypercholesterolemia and atherosclerosis, these important functions of the vascular endothelium are strongly impaired. One of the major reasons for this so-called endothelial dysfunction is the increased vascular generation of reactive oxygen species such as superoxide and peroxynitrite. We aimed to investigate whether superoxide and peroxynitrite impacts on the expression and function of sGC and if such a mechanism occurs in a hypercholestemia-induced atherosclerosis. Our experiments with isolated rat aortic rings showed that extracellular superoxide has no effect on expression and function of sGC, while subjection of these rings to continuously generated extracellular peroxynitrite reduced sGC activity. Furthermore, intracellular superoxide as generated by LY85385 almost completely inhibited sGC-activity and increased its expression. In the cholesterol-fed White New Zealand rabbit, we found a 3.5-fold upregulation of sGC, while basal and NO-stimulated sGC-activities were only slightly enhanced and the vasodilator potency of SNAP was decreased by 10-fold. A great portion of the overexpressed dysfunctional sGC is located in intimal lesions. Finally, platelet sGC-activity and the anti-aggregatory effect of SNAP were not changed. These data suggest that endothelial dysfunction in hypercholesterolemia is associated with an oxidative stress-dependent and reversible overexpression of a dysfunctional vascular sGC, while inhibition of platelet sGC-activity is most likely not involved in hypercholesterolemia-induced platelet hyperreactivity.

Topics: Animals; Blood Platelets; Blood Vessels; Cyclic GMP; Guanylate Cyclase; Humans; Hypercholesterolemia; Nitric Oxide; Oxidative Stress; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase

Upon sexual stimulation, penile erection, occurring in response to the activation of pro-erectile autonomic pathways, is greatly dependent on adequate inflow of blood to the erectile tissue and requires coordinated arterial endothelium-dependent vasodilatation and sinusoidal endothelium-dependent corporal smooth muscle relaxation. Nitric oxide (NO) is the principal peripheral pro-erectile neurotransmitter which is released by both non-adrenergic, non-cholinergic neurons and the sinusoidal endothelium to relax corporal smooth muscle through the cGMP pathway. Any factors modifying the basal corporal tone, the arterial inflow of blood to the corpora, the synthesis/release of neurogenic or endothelial NO are prime suspects for being involved in the pathophysiology of erectile dysfunction (ED). In fact, conditions associated with altered endothelial function, such as ageing, hypertension, hypercholesterolemia and diabetes, may, by changing the balance between contractant and relaxant factors, cause circulatory and structural changes in penile tissues, resulting in arterial insufficiency and defect in smooth muscle relaxation and thus, ED. There is increasing evidence to suggest that ED is predominantly a vascular disease and may even be a marker for occult cardiovascular disease. Recent results illustrating the importance of endothelial dysfunction in the pathophysiology of different forms of experimental ED are discussed. These pathways may represent new potential treatment targets.

Topics: Animals; Cardiovascular Diseases; Cyclic GMP; Diabetes Complications; Diabetes Mellitus, Experimental; Endothelium, Vascular; Erectile Dysfunction; Humans; Hypercholesterolemia; Hypertension; Male; Models, Biological; Muscle, Smooth, Vascular; Nitric Oxide; Penile Erection; Rabbits; Rats; Rats, Inbred SHR; Risk Factors; Vasoconstriction; Vasodilation

This review focuses on the role of impaired endothelial function for the development of atherosclerosis in human arterial hypertension and hypercholesterolemia in vivo. Potential mechanisms underlying impaired endothelial function and decreased bioavailability of nitric oxide under these clinical conditions are discussed. It further addresses therapeutic strategies aimed at improving the bioavailability of nitric oxide in these patients. The overall conclusion is that the bioavailability of nitric oxide is probably impaired, not by a single defect, but by various mechanisms affecting nitric oxide synthesis as well as nitric oxide breakdown. In both diseases increased superoxide anion production and oxidative stress represent a major mechanism. Decreased bioavailability of nitric oxide not only impairs endothelium-dependent vasodilation, but also activates other mechanisms that play an important role in the pathogenesis of atherosclerosis. Thus, therapeutic strategies should aim to restore bioavailability of nitric oxide, which has been demonstrated for lipid-lowering therapy in hypercholesterolemia and blood pressure control in hypertension. In addition, antioxidative strategies will represent a major therapeutic tool against atherosclerotic diseases in the future. Statins and blockers of the renin-angiotensin system seem to have such antioxidative effects independent from their effects on lipid profiles or blood pressure control.

Topics: Animals; Arginine; Biological Availability; Cyclic GMP; Endothelium, Vascular; Humans; Hypercholesterolemia; Hypertension; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Prognosis; Vasodilation

Topics: Animals; Biological Factors; Cyclic GMP; Diabetes Mellitus; Endothelium, Vascular; Epoprostenol; Humans; Hypercholesterolemia; Hypertension; Muscle, Smooth, Vascular; Nitric Oxide; Vasodilation

Anthocyanins have been shown to improve endothelial function in animal models. However, whether these compounds have similar beneficial effects in humans is largely unknown.. In a short-term crossover study, 12 hypercholesterolemic individuals were given oral anthocyanins (320 mg) isolated from berries or placebo. Brachial artery flow-mediated dilation (FMD) was assessed before and after the intervention. In a long-term intervention trial (12 weeks), 150 hypercholesterolemic individuals were given anthocyanins (320 mg/day, n = 75) or placebo (n = 75), after which we measured FMD, plasma cGMP, and other serum biomarkers. Another short-term intervention was conducted in the presence of NO-cGMP inhibitors in 6 people and in a rat aortic ring model (n = 8).. Significant increases of FMD from 8.3% (0.6%) at baseline to 11.0% (0.8%) at 1 h and 10.1% (0.9%) at 2 h were observed after short-term anthocyanin consumption, concomitantly with increases of plasma anthocyanin concentrations (P < 0.05). In the study participants who received long-term anthocyanin intervention, compared with the control group, we observed significant increases in the FMD (28.4% vs 2.2%), cGMP (12.6% vs -1.2%), and HDL-cholesterol concentrations, but decreases in the serum soluble vascular adhesion molecule-1 and LDL cholesterol concentrations (P < 0.05). The changes in the cGMP and HDL cholesterol concentrations positively correlated with FMD in the anthocyanin group (P < 0.05). In the presence of NO-cGMP inhibitors, the effects of anthocyanin on endothelial function were abolished in human participants and in a rat aortic ring model.. Anthocyanin supplementation improves endothelium-dependent vasodilation in hypercholesterolemic individuals. This effect involves activation of the NO-cGMP signaling pathway, improvements in the serum lipid profile, and decreased inflammation.

Topics: Adult; Aged; Animals; Anthocyanins; Aorta, Thoracic; Body Weights and Measures; Brachial Artery; Cholesterol, HDL; Cholesterol, LDL; Cross-Over Studies; Cyclic GMP; Dietary Supplements; Endothelium, Vascular; Humans; Hypercholesterolemia; In Vitro Techniques; Male; Middle Aged; Rats; Ribes; Time Factors; Vaccinium myrtillus; Vascular Cell Adhesion Molecule-1; Vasodilation

We previously demonstrated that normotensive patients with hypercholesterolemia showed excessive blood pressure (BP) responses to stress tests. In this study, we examined the effects of cholesterol-lowering therapy on BP in order to confirm that hypercholesterolemia plays a role in the regulation of BP. Fifteen patients with hypercholesterolemia and 24 normal cholesterolemic controls performed mental arithmetic stress (AS) and hand grip (HG) tests. BP was measured during the tests. Serum lipids and lipid peroxides were measured before the AS. Platelet intracellular free calcium concentration ([Ca2+])i with and without low density lipoprotein (LDL) stimulation, plasma cGMP and NOx were determined immediately before AS and at the end of each test. In hypercholesterolemic patients, the tests were repeated at the end of a 12-week treatment with 10 mg/day of pravastatin, a hepatic hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. In hypercholesterolemic patients, BP responses to both tests were greater than those of the controls. Basal and LDL-stimulated platelet [Ca2+]i were higher, and the ratio of plasma cGMP to NOx was lower. Serum total cholesterol, LDL cholesterol and lipid peroxides were significantly decreased in association with the pravastatin treatment. Systolic BP to AS and systolic BP/diastolic BP to HG were decreased (p < 0.01, p < 0.01/p < 0.02, respectively). Platelet [Ca2+]i with LDL stimulation was decreased (p < 0.01). Plasma cGMP was increased (p < 0.05), whereas NOx was decreased (p < 0.05); therefore, the ratio of cGMP to NOx was increased (p < 0.05). In conclusion, excessive blood pressure responses to stress tests were improved after cholesterol-lowering therapy. This finding suggests that hypercholesterolemia itself is involved in the regulation of BP.

Topics: Anticholesteremic Agents; Blood Platelets; Blood Pressure; Calcium; Catecholamines; Cyclic GMP; Female; Hand Strength; Heart Rate; Humans; Hypercholesterolemia; Lipid Peroxides; Lipids; Male; Middle Aged; Nitric Oxide; Pravastatin; Stress, Psychological

Hypercholesterolaemia has been found to impair endothelial function in the systemic and coronary circulations and lipid-lowering therapy with statins has been shown to improve this abnormality.. We examined the impact of hypercholesterolaemia on L-arginine-induced renal vascular relaxation by a cross-sectional study, and the effects of lipid-lowering therapy by a double-blind, randomized, placebo-controlled study. Using constant infusion input clearance technique (PAH and inulin respectively), changes of renal plasma flow (RPF) and glomerular filtration rate (GFR) in response to intravenous infusions of L-arginine (100 mg/kg/30 min and 500 mg/kg/30 min) were studied in 21 hypercholesterolaemic humans (age 57+/-9 years, LDL-cholesterol 211+/-35 mg/dl) and in 20 young healthy (age 26+/-2 years, LDL-cholesterol 90+/-27 mg/dl) and 20 older healthy age-matched control individuals (age 50+/-8 years, LDL-cholesterol 106+/-20 mg/dl). In addition, changes of blood pressure, heart rate, urinary excretion of sodium, and cyclic guanosine monophosphate were measured. Patients were analysed before and after 3 months treatment with either fluvastatin (40 mg twice daily, n=11) or placebo (n=10).. In hypercholesterolaemic patients, L-arginine increased RPF and GFR (P<0.01) and urinary excretion of sodium (P<0.05) in a dose-dependent manner. Interestingly, changes were similar between the hypercholesterolaemic patients and the young and the age-matched control individuals (DeltaRPF 100 mg/kg/30 min, 40+/-51 ml/min vs 40+/-52 ml/min, P=NS; DeltaRPF 500 mg/kg/30 min, 114+/-85 ml/min vs 130+/-78 ml/min, P=NS). L-arginine significantly lowered systemic arterial pressure and increased heart rate in all groups. Despite significant reductions in LDL-cholesterol levels (291+/-35 mg/dl vs 213+/-30 mg/dl, P<0.001), treatment with fluvastatin did not alter the renal haemodynamic response pattern to L-arginine infusion when compared to baseline values and to those with placebo.. In contrast to studies performed in the vasculature of the human forearm or the coronary circulation, our results suggest that hypercholesterolaemia is not associated with an impaired L-arginine-induced renal vascular relaxation.

Topics: Aged; Arginine; Blood Pressure; Cohort Studies; Cyclic GMP; Double-Blind Method; Fatty Acids, Monounsaturated; Female; Fluvastatin; Heart Rate; Hemodynamics; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; Indoles; Male; Middle Aged; Natriuresis; Osmolar Concentration; Renal Circulation; Vasodilation; Vasodilator Agents

The short-term effects of hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) on endothelial function at doses that do not affect plasma lipid levels are not known.. We investigated the short-term effects of cerivastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, on endothelial function and endothelium-related products in elderly diabetic patients. Twenty-seven elderly diabetic patients (aged 69.3+/-3.4 years), with or without mild hypercholesterolemia, were enrolled in this study, which tested cerivastatin treatment (0.15 mg/d) for 3 days. Endothelium-dependent flow-mediated dilatation, endothelium-independent dilatation by nitroglycerin in the brachial artery, nitric oxide-related products (nitrite/nitrate and cGMP), endothelium-related products (von Willebrand Factor, soluble vascular cell adhesion molecule-1, and soluble intercellular adhesion molecule-1), and a marker of oxidant stress (8-isoprostane) were assessed. Levels of plasma lipids were not changed before and after treatment with cerivastatin. Flow-mediated dilatation was significantly increased by cerivastatin treatment, as were plasma nitrite/nitrate levels (from 16.9+/-3.4 to 22.0+/-3.7 micromol/L, P<0.05) and cGMP values. The percent of nitroglycerin-induced dilatation was not changed. Plasma concentrations of 8-isoprostane decreased, and levels of soluble vascular cell adhesion molecule also tended to decrease with cerivastatin.. Improvement of endothelial function was in line with antiatherosclerotic effects. Cerivastatin improved impaired endothelial function in the short-term without affecting lipid profiles in elderly diabetic patients. This effect may be partly due to upregulation of endothelial nitric oxide synthase.

Topics: Aged; Brachial Artery; Cyclic GMP; Diabetes Complications; Diabetes Mellitus; Endothelium, Vascular; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; Lipids; Male; Nitrates; Nitrites; Pyridines; Time Factors; Treatment Outcome; Vasodilation

Despite the advent of oral phosphodiesterase-5 inhibitors, curative treatment for erectile dysfunction (ED) remains unavailable. Recently, the link between ED and cardiovascular disease was unveiled and the main etiology of ED was found to be vasculogenic. Therefore, neovascularization is a promising strategy for curing ED. Angiopoietin-1 (Ang1) is an angiogenic growth factor that promotes the generation of stable and functional vasculature. Here, we demonstrate that local delivery of the soluble, stable, and potent Ang1 variant, COMP-Ang1 gene or protein, into the penises of hypercholesterolemic mice increases cavernous angiogenesis, eNOS phosphorylation, and cGMP expression, resulting in full recovery of erectile function and cavernous blood flow up to 8 weeks after treatment. COMP-Ang1-induced promotion of cavernous angiogenesis and erectile function was abolished in Nos3(-/-) mice and in the presence of the NOS inhibitor, L-NAME. COMP-Ang1 also restored the integrity of endothelial cell-cell junction by down-regulating the expression of histone deacetylase 2 in the penis of hypercholesterolemic mice and in primary cultured mouse cavernous endothelial cells. These findings constitute a new paradigm toward curative treatment of both cavernous angiopathy and ED.

Topics: Animals; Cells, Cultured; Cyclic GMP; Diet, High-Fat; Disease Models, Animal; Down-Regulation; Endothelial Cells; Histone Deacetylase 2; Hypercholesterolemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Pathologic; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Penile Erection; Penis; Phosphorylation; Recombinant Fusion Proteins; Regional Blood Flow; Tight Junction Proteins

The mechanisms by which heme oxygenase (HO) improves glucose metabolism in essential hypertension are not completely understood. Because dysfunctional insulin signaling is associated with elevated inflammation and high cholesterol and triglycerides, we investigated the effects of HO on the proinflammatory macrophage M1 phenotype and the anti-inflammatory macrophage M2 phenotype in spontaneously hypertensive rats (SHRs). SHRs are a model of human essential hypertension with features of metabolic syndrome, including impaired glucose metabolism.. Spectrophotometric analysis, enzyme immunoassay, enzyme-linked immunosorbent assay, and Western immunoblotting were used. HO was enhanced with hemin or inhibited with chromium-mesoporphyrin (CrMP).. Hemin suppressed inflammation by abating proinflammatory macro phage M1 phenotype (ED1) and chemokines such as macrophage chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 alpha (MIP-1α) while enhancing anti-inflammatory macrophage M2 phenotype by potentiating ED2, CD206, and CD14. Similarly, hemin improved insulin signaling by enhancing insulin receptor substrate 1 (IRS-1), IRS-2, phosphatidylinositol 3 kinase (PI3K), and glucose transporter 4 (GLUT4) but reduced total cholesterol and triglycerides. These effects were accompanied by increased HO-1, HO activity, and cyclic guanosine monophosphate (cGMP), whereas the HO inhibitor CrMP nullified the hemin effects. Importantly, the effects of the HO system on ED1, ED2, CD206, and CD14 in SHRs are novel.. Hemin abated inflammation in SHRs by selectively enhancing the anti-inflammatory macrophage M2 phenotype that dampens inflammation while suppressing the pronflammatory macrophage M1 phenotype and related chemokines such as MCP-1 and MIP-1α. Importantly, the reduction of inflammation, total cholesterol, and triglycerides was accompanied by the enhancement of important proteins implicated in insulin signaling, including IRS-1, IRS-2, PI3K, and GLUT4. Thus, the concomitant reduction of inflammation, total cholesterol and triglycerides and the corresponding potentiation of insulin signaling are among the multifaceted mechanisms by which the HO system improves glucose metabolism in essential hypertension.

Topics: Animals; Blood Glucose; Blood Pressure; Chemokine CCL2; Chemokine CCL3; Cyclic GMP; Essential Hypertension; Heme Oxygenase (Decyclizing); Hemin; Hypercholesterolemia; Hypertension; Hypertriglyceridemia; Insulin; Liver; Macrophages; Male; Mesoporphyrins; Muscle, Skeletal; Phenotype; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Signal Transduction

Hypercholesterolemia is associated with decreased nitric oxide (NO) bioavailability and endothelial dysfunction, a phenomenon thought to have a major role in the altered cerebral blood flow evident in stroke. Therefore, strategies that increase endothelial NO production have potential utility. Vascular reactivity of the middle cerebral artery (MCA) from C57BL/6J wild-type (WT) mice, apolipoprotein-E knockout (ApoE(-/-)) mice, and mice treated with the phosphodiesterase inhibitor cilostazol (100 mg/kg) was analyzed using the tension myograph. Contractile responses to endothelin-1 were significantly enhanced in MCA from ApoE(-/-) mice compared with WT mice (P<0.01), an effect absent in cilostazol-treated ApoE(-/-) mice. Acetylcholine-induced relaxation (which is entirely NO-dependent) was significantly impaired in MCA of ApoE(-/-) mice compared with WT mice (P<0.05), again an effect prevented by cilostazol treatment. Endothelial NOS phosphorylation at Ser(1179) was decreased in the aorta of ApoE(-/-) mice compared with WT mice (P<0.05), an effect normalized by cilostazol. Taken together, our data suggest that the endothelial dysfunction observed in MCA associated with hypercholesterolemia is prevented by cilostazol, an effect likely due to the increase in eNOS phosphorylation and, therefore, activity.

Topics: Animals; Aorta; Apolipoproteins E; Cells, Cultured; Cilostazol; Cyclic GMP; Endothelin-1; Endothelium, Vascular; Humans; Hypercholesterolemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Cerebral Artery; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Phosphodiesterase Inhibitors; Tetrazoles; Vasoconstriction; Vasodilation

Studies using cultured endothelial cells have shown that high-density lipoprotein (HDL) positively modulates endothelial nitric oxide synthase (eNOS). The purpose of this study was to test the hypotheses that positive modulation of eNOS by HDL occurs in whole vessels and that it augments endothelium-dependent vasorelaxation. To test these hypotheses, brachial arteries were obtained from swine. Endothelium-dependent and endothelium-independent vasorelaxation were determined in vitro to assess the effects of acute administration of HDL (50 microg.mL-1; n = 8) and chronic exposure to relatively high HDL concentration on vascular function (low HDL, 0.89 +/- 0.02 mmol.L-1, n = 4; high HDL, 1.16 +/- 0.05 mmol.L-1, n = 4; p < 0.005). Acute administration of HDL did not augment maximal endothelium-dependent vasorelaxation to bradykinin (BK) (no HDL, 82.6% +/- 2.2%; HDL, 76.7% +/- 3.5%; not significant (ns)). Similarly, maximal relaxation to BK was not enhanced by chronic exposure to high HDL concentrations. NO synthase (NOS) activity was also similar between groups (low HDL, 129.0 +/- 19.2 counts.h-1.microg-1 protein; high HDL, 113.9 +/- 47.1 counts.h-1.microg-1; ns). Consistent with NOS activity, the extent of eNOS phosphorylation at several sites was similar between low HDL and high HDL. Both apolipoprotein A-I (ApoA-I) and scavenger receptor class B type I (SR-BI) were associated with eNOS. Similar to cultured cell studies, this study demonstrates that both ApoA-I and SR-BI associate with eNOS in the vascular wall. Binding of ApoA-I and SR-BI to eNOS does not, however, result in modulation of either NO formation or endothelial function.

Topics: Animals; Apolipoprotein A-I; Brachial Artery; Bradykinin; CD36 Antigens; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Hypercholesterolemia; Lipoproteins, HDL; Nitric Oxide Synthase Type III; Nitroprusside; Phosphorylation; Swine; Vasodilation; Vasodilator Agents

Endothelial function is impaired in hypercholesterolemia and atherosclerosis. Based on mostly indirect evidence, this impairment is attributed to reduced synthesis or impaired biological activity of endothelium-derived nitric oxide (NO). It was the aim of this study to directly estimate and compare whole-body NO production in normo- and hypercholesterolemia by applying a nonradioactive stable isotope dilution technique in vivo.. We enrolled 12 normocholesterolemic and 24 hypercholesterolemic volunteers who were all clinically healthy. To assess whole-body NO synthesis, we intravenously administered l-[guanidino-((15)N(2))]-arginine and determined the urinary excretion of (15)N-labeled nitrate, the specific end product of NO oxidation in humans, by use of gas chromatography-mass spectrometry. In addition, we measured flow-mediated vasodilation (FMD) of the brachial artery, expression of endothelial NOS (eNOS) in platelets, plasma concentration of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), and urinary excretion of 8-isoprostaglandin F(2alpha) (8-iso-PGF(2alpha)).. After infusion of l-[guanidino-((15)N(2))]-arginine, cumulative excretion of (15)N-labeled-nitrate during 48 h was 40% [95% CI 15%-66%] lower in hypercholesterolemic than normocholesterolemic volunteers [mean 9.2 (SE 0.8) micromol vs 15.4 (2.3) micromol/l, P = 0.003]. FMD was on average 36% [4%-67%] lower in hypercholesterolemic than normocholesterolemic volunteers [6.3 (4.0)% vs 9.4 (4.6)%, P = 0.027]. Normalized expression of NOS protein in platelets was also significantly lower in hypercholesterolemic volunteers, whereas there were no significant differences in plasma ADMA concentration or urinary excretion of 8-iso-PGF(2alpha) between the 2 groups.. This study provides direct evidence for a decreased whole body NO synthesis rate in healthy people with hypercholesterolemia.

Topics: Adolescent; Adult; Aged; Arginine; Cyclic GMP; Dinoprost; Endothelium, Vascular; Female; Humans; Hypercholesterolemia; Male; Middle Aged; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidative Stress; Sex Factors

Hypercholesterolemia is one of the most important risk factors for the development of erectile dysfunction (ED) in men.. We employed an established mouse model of hypercholesterolemia.. We test for abnormalities in vasoreactivity in corporal tissue and temporally correlated changes in vasoreactivity with alterations in histology and protein expression.. A total of 150 mice were studied. A total of 100 apolipoprotein-E knockout (ApoE(-/-)) mice were fed a 1.25% cholesterol diet for 2, 4, 8, and 12 weeks (N = 25/group), while a group of ApoE(-/-) and wild-type Bl-6 mice were fed a normal diet. The study was terminated, and all mice were harvested at 22 weeks of age for vasoreactivity, histology, and protein studies from corporal tissues. Dose-response curves were generated to evaluate endothelium-dependent and endothelium-independent vasoreactivity, ex vivo. The contents of endothelial cells, smooth muscle cells, and smooth muscle/collagen ratio were assessed by immunohistochemistry staining or Masson staining. Level of cyclic guanosine monophosphate (cGMP) was detected by enzyme immunoassay assay. Levels of phosphorylated endothelial nitric oxide synthase (p-eNOS)/total eNOS, neuronal nitric oxide synthase (nNOS), and cyclic GMP-dependent kinase (cGK-1) protein were assessed by Western analysis.. Abnormalities in endothelium-dependent and endothelium-independent vasoreactivities, endothelial content, smooth muscle/collagen ratio, p-eNOS phosphorylation at Ser1177 only, nNOS, cGMP, and cGK-1 changed with the different durations of the high-cholesterol diet.. These data demonstrate that this mouse model is suitable for investigating aspects of hypercholesterolemic ED.

Topics: Animals; Blotting, Western; Cholesterol; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Erectile Dysfunction; Guanosine Monophosphate; Hypercholesterolemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Penis

Endothelial dysfunction is characterized by impaired vasodilation, increase of oxidative stress and inflammation. The current study was designed to test the hypothesis that reversal of hypercholesterolemic diet alone does not normalize all the parameters of endothelial dysfunction. After 10 weeks on a high-cholesterol diet, female juvenile pigs were randomized to normal diet (n=5, "Reversals") or continued on the same diet (n=6, "HC") for another 6 weeks. A control group of 11 pigs received a normal diet ("C"). Coronary epicardial and arteriolar endothelial function was tested in vitro. NFkappaB and p47phox expression was analyzed in epicardial arteries and myocardium, respectively. P47phox localization in coronary arteries was demonstrated with immunohistochemistry. Lipid levels normalized in Reversal pigs. Epicardial arteries of Reversals showed a normalized relaxation and NFkappaB expression compared to HC (p<0.05). Small vessel relaxation remained attenuated, and expression of p47phox in myocardial tissue was elevated in Reversals compared to C (p<0.05). Dietary lowering of serum cholesterol and LDL improves vascular function of epicardial arteries but neither of small vessels nor vascular oxidative stress within this time frame. Hence, dietary normalization of serum lipid levels alone may not be synonymous to normalization of the components of endothelial dysfunction.

Topics: Analysis of Variance; Animals; Blotting, Western; Coronary Vessels; Cyclic GMP; Diet, Atherogenic; Endothelium, Vascular; Female; Hypercholesterolemia; Immunohistochemistry; NADPH Oxidases; NF-kappa B; Superoxide Dismutase; Sus scrofa

Our previous studies have proven that crocetin (CCT), extracted from Gardenia jasminoides Ellis, possesses the anti-atherosclerotic effect. Because endothelial dysfunction strongly contributes to the initiation and progression of atherosclerosis, the present study aims to investigate whether CCT is capable of improving this dysfunction and to explore the possible mechanisms. Endothelial dysfunction was induced by in vivo feeding high cholesterol diet (HCD) to rabbit and by in vitro treating bovine aortic endothelial cells (BAECs) with oxidized LDL (oxLDL). Endothelium-dependent relaxation (EDR) evoked by acetylcholine (Ach) and endothelium-independent relaxation (RIDR) mediated by sodium nitroprusside (SNP) of thoracic aorta isolated from rabbit were measured. The results indicated that the EDR in HCD alone treated rabbits was seriously impaired and the maximal relaxation induced by Ach (10(-5.5) M) was only 54% that in control rabbit fed with regular diet. Oral complementation with CCT (15, 30 mg/kg) dose-dependently improved this impairment and restored the maximal relaxation to 68% and 80% that in control group, respectively. However, the EIDR maintained comparable in all groups. Complementation with CCT (15, 30 mg/kg) simultaneously increased serum level of nitric oxide (NO), upregulated vessel activity and mRNA expression of endothelial NO synthase (eNOS) as well as vessel cyclic GMP (cGMP) content compared with those in rabbit treated with HCD alone. Inducible NOS (iNOS) activity remained unchangeable in all groups. In BAECs, oxLDL treatment decreased NO production, downregulated both activity and mRNA expression of eNOS. While those decrease or downregulation were inhibited by co-treatment with CCT (0.1, 1, 10 microM) in a dose-dependent manner. These findings suggested that CCT significantly restored the EDR of thoracic aorta in hypercholesterolemic rabbit, which might be explained by its action to increase the vessel eNOS activity, leading to elevation of NO production.

Topics: Animals; Aorta, Abdominal; Aorta, Thoracic; Base Sequence; Carotenoids; Cyclic GMP; DNA Primers; Endothelium, Vascular; Hypercholesterolemia; Male; Muscle Relaxation; Nitric Oxide; Nitric Oxide Synthase Type III; Rabbits; RNA, Messenger; Vitamin A

The endogenous insulin sensitizing machinery termed the hepatic insulin sensitizing substance (HISS) mechanism has been shown to be nitrergic and linked to sensory fibers in the anterior hepatic plexus. We studied whether this mechanism could pharmacologically be exploited by cicletanine, a cGMP-PDE inhibitor antihypertensive drug, in conscious rabbits. Whole body insulin sensitivity and peripheral glucose uptake were determined by hyperinsulinaemic euglycaemic glucose clamping, and cardiac radiolabelled deoxyglucose (DOG) uptake in neurogenic, achieved by perineurial capsaicin treatment of the anterior hepatic plexus through defunctionalization of hepatic sensory fibers, and metabolic, induced by dietary hypercholesterolemia, insulin resistance models after single oral doses of cicletanine (3, 10 and 30 mg kg(-1)) or rosiglitazone (3 mg kg(-1)). The effect of cicletanine on cardiac and vascular tissue NO, cGMP, cAMP was measured by means of spin trapping technique and radioimmunoassay, respectively. Insulin sensitivity and peripheral DOG uptake were significantly increased by 10 and 30 mg kg(-1) cicletanine in both healthy and hypercholesterolaemic rabbits, but not in those with neurogenic insulin resistance. Rosiglitazone had no effect in healthy and neurogenic insulin resistant rabbits although it improved insulin sensitivity in hypercholesterolemic animals. The 10 mg kg(-1) cicletanine dose induced no change in either cardiac or vascular tissue NO, cGMP or cAMP concentrations. Nevertheless, at a dose of 30 mg kg(-1) producing an insulin sensitizing effect of approximately the same amplitude as seen with 10 mg kg(-1), the drug significantly increased tissue NO and cGMP concentrations. Oral cicletanine attains its insulin sensitizing effect at doses lower than those necessary to activate the NO-cGMP pathway in the cardiovascular system. This metabolic effect requires functional integrity of hepatic sensory nerves.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blood Pressure; Blood Vessels; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glucose; Glucose Clamp Technique; Heart Rate; Hypercholesterolemia; Hyperinsulinism; Hypoglycemic Agents; Insulin Resistance; Lipids; Male; Myocardium; Nitric Oxide; Pyridines; Rabbits; Rosiglitazone; Thiazolidinediones

The pathophysiological effects of the activation or inhibition of the nitric oxide (NO)-mediated pathway on the deformability of red blood cells (RBC) were evaluated in the presence of hypercholesterolemia induced in rabbits fed a cholesterol-rich diet. RBC deformability was assessed using a microchannel array flow analyzer system. The maximum passage time (MPT) by flowing a suspension of RBC through the microchannels was used as an index of RBC deformability. During cholesterol feeding for 12 weeks, MPT gradually increased with no significant elevation in the serum asymmetric dimethylarginine (ADMA) and arginine/ADMA ratio. The reduction in RBC deformability associated with hypercholesterolemia was significantly improved during incubation with each of three different NO pathway activators: a NO donor, 8-bromo-cyclic GMP, and arginine; however, no additional reduction was observed with ADMA administration. The inhibition of NO synthase due to ADMA caused a significant reduction in the deformability of normal RBC, which was reversed with NO pathway activation. These results suggest that impaired RBC deformability may be associated with a dysfunction in the NO pathway that is partially dependent upon the accumulation of ADMA in RBC, and exogenous NO pathway activators may improve the microcirculation by restoring RBC deformability in the presence of hypercholesterolemia.

Topics: Animals; Arginine; Blood Chemical Analysis; Cyclic GMP; Enzyme Inhibitors; Erythrocytes; Female; Hypercholesterolemia; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Rabbits

Hypercholesterolemia impairs endothelial function. However, the critical level of serum total cholesterol at which endothelial dysfunction occurs is unknown at present. We investigated cross-sectionally the correlation between urinary excretion of cyclic guanosine 3',5' monophosphate (cGMP), a second messenger of nitric oxide (NO) and serum total cholesterol concentrations in a general population sample of Japanese men and women. The samples comprised 1541 subjects (788 men and 753 women) aged 40-79 years, who participated in cardiovascular risk surveys between 1997 and 2002 and underwent a 24h urine collection. Urinary excretion of cGMP was measured using a (125)I-labeled cGMP radioimmunoassay and was adjusted for urinary creatinine excretion (nmol/mmol creatinine). The mean urinary cGMP excretion correlated linearly and inversely with serum total cholesterol level: mean cGMP excretion adjusted for age, sex and cardiovascular risk factors was 61.7, 53.6, 50.8, 49.2, 47.3 and 46.4 nmol/mmol for total cholesterol levels <4.14, 4.14-4.64, 4.65-5.16, 5.17-5.68, 5.69-6.20 and > or =6.21 mmol/L, respectively (p=0.007). This relation was more evident among individuals with end-organ damage, among subjects with higher C-reactive protein (CRP) levels and among postmenopausal women. Our data suggest a reduction of NO bioactivity with higher serum total cholesterol levels, even within clinically normal cholesterol levels.

Topics: Adult; Age Factors; Aged; Cholesterol; Cross-Sectional Studies; Cyclic GMP; Female; Humans; Hypercholesterolemia; Male; Menopause; Middle Aged; Nitric Oxide; Reference Values; Second Messenger Systems; Sex Factors

Chronic exposure to L-arginine results in regression of atherosclerotic lesions and reversal of endothelial dysfunction. We investigated whether chronic L-arginine supplementation induces regression of atherosclerotic lesions and reversal of endothelial dysfunction in atherogenic rhesus monkeys and the mechanism which leads to these effects. About 12 male rhesus monkeys were fed 1% cholesterol and 18 g butter for 6 months to create an experimental model of hypercholesterolaemia and atherosclerosis (Group I) and 12 monkeys were fed standard stock diet for 6 months (Group II). After, 6 months these two groups were further divided into 2 sub-groups which in addition to their respective diets were fed 2.5% L-arginine in drinking water for additional 6 months (Group III and Group IV). Systemic nitric oxide (NO) formation was assessed as plasma nitrite and cGMP formation every 3 months. Oxygen free radical (OFR) generation and malondialdehyde production as an index of lipid peroxidation were determined. Changes in isometric tension were compared in isolated ring segments of thoracic aorta from normal and hypercholesterolemic animals. Cholesterol feeding progressively reduced plasma nitrite and cGMP generation (p < 0.05). Dietary L-arginine partly restored the levels of plasma nitrite and cGMP (p < 0.05) but did not change plasma cholesterol levels. L-arginine significantly reduced aortic intimal thickening, blocked the production of carotid and coronary intimal plaques and completely preserved endothelium-dependent vasodilator function. Further, L-arginine significantly inhibited generation of the reactive oxygen species (ROS) generation and lipid peroxidation. Chronic oral supplementation with L-arginine blocks the progression of plaques via restoration of nitric oxide synthase substrate availability and reduction of vascular oxidative stress.

Topics: Animals; Arginine; Cholesterol, Dietary; Coronary Artery Disease; Cyclic GMP; Diet, Atherogenic; Endothelium, Vascular; Hypercholesterolemia; Lipid Peroxidation; Macaca mulatta; Male; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Superoxides

Hypercholesterolaemia promotes erectile dysfunction through increased superoxide formation and negation of nitric oxide (NO) bioactivity in cavernosal tissue. The source of superoxide has not been clearly defined, however. Sildenafil (Viagra), the standard therapy for erectile dysfunction, may also be rendered more effective by the presence of an NO donor. One drug that intrinsically fulfils this criterion is sildenafil nitrate (NCX 911), an NO donating derivative of sildenafil. The objective of this study, therefore, was to determine the source of superoxide and its effect on erectile function in corpus cavernosum from hypercholesterolaemic rabbits and to determine whether NCX 911 confers an improvement over sildenafil citrate in this model. Hypercholesterolaemia elicited an increase in superoxide formation by rabbit cavernosal tissue and a reduction of carbachol-stimulated relaxation both of which were reversed by diphenylene iodonium chloride and apocynin (NADPH oxidase inhibitors). In response to sodium nitroprusside, hypercholesterolaemia also caused an attenuation of cavernosal relaxation which was not reversed with NADPH oxidase inhibitors. Both sildenafil citrate and NCX 911 significantly reversed impaired carbachol-stimulated relaxation and inhibited superoxide formation by cavernosal tissue from hypercholesterolaemic rabbits, NCX 911 being more potent. NCX 911 also augmented cavernosal cGMP levels, an effect blocked by the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo {4,3-a}quinoxalin-1-one (ODQ). These data demonstrate that hypercholesterolaemia promotes erectile dysfunction through an augmentation of superoxide derived from NADPH oxidase in cavernosal tissue. It also indicates that NO donating sildenafil may be therapeutically more beneficial than conventional sildenafil in treating erectile dysfunction with an oxidative stress-related aetiology.

Topics: Acetophenones; Allopurinol; Animals; Carbachol; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypercholesterolemia; In Vitro Techniques; Male; Muscle Relaxation; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Onium Compounds; Oxadiazoles; Penis; Phosphodiesterase Inhibitors; Piperazines; Purines; Quinoxalines; Rabbits; Rotenone; Sildenafil Citrate; Sulfones; Superoxides; Uncoupling Agents; Xanthine Oxidase

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Electric Stimulation Therapy; Hypercholesterolemia; Male; Penile Erection; Penis; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases

Coronary endothelial dysfunction is associated with an increase in cardiac events. Hypercholesterolemia (HC) and hypertension (HT) are both associated with endothelial dysfunction, and their coexistence is associated with an increased incidence of cardiac events in epidemiological studies. However, pathogenic mechanisms are poorly understood. Here we studied the effects of coexisting HC and HT on coronary endothelial function.. Four groups of pigs were studied after 12 weeks of a normal diet (n=9), a 2% HC diet (n=9), HT (achieved by unilateral renal artery stenosis, n=8), or HC+HT (n=6). Coronary endothelial function was tested, in epicardial arteries and arterioles, by using organ chamber techniques. Oxidative stress was measured in coronary artery tissue. Vasodilatory response to bradykinin and calcium ionophore was significantly impaired in animals with HC+HT compared with each risk factor alone (P<0.05 for both). In animals with coexistent HC and HT, the increase in oxidative stress was more pronounced compared with each risk factor alone (P<0.05). Furthermore, chronic antioxidant supplementation significantly improved coronary artery vasoreactivity.. These results suggest that HC and HT have a synergistic deleterious effect on coronary endothelial function, associated with increased oxidative stress. This interaction may contribute to the increased incidence of coronary heart disease and cardiac events seen when HC and HT coexist.

Topics: Animals; Antioxidants; Ascorbic Acid; Bradykinin; Calcimycin; Coronary Artery Disease; Coronary Vessels; Cyclic GMP; Diet, Atherogenic; Endothelin-1; Endothelium, Vascular; Female; Hemodynamics; Hypercholesterolemia; Hypertension, Renovascular; Lipids; Nitric Oxide; Nitroprusside; Oxidative Stress; Renal Artery Obstruction; Renin; Substance P; Swine; Vasodilator Agents; Vitamin E

Although sarpogrelate HCl is widely used for the prevention of arterial thrombosis, its effect on atherosclerosis is unknown. Accordingly, we here investigated the effects of sarpogrelate HCl on a rabbit model of atherosclerosis. Male rabbits were fed a 0.5% cholesterol diet (HCD) (Gp 1), HCD with vitamin E (Gp 2), HCD with vitamin E and sarpogrelate (Gp 3), or HCD with sarpogrelate alone (Gp 4) for 8 weeks. The atherosclerotic area was decreased by feeding of vitamin E and sarpogrelate (16.9+/-2.0% in Gp 1 vs. 8.2+/-2.0% in Gp 3). Tone-related basal NO release was higher in Gps 3 and 4. Acetylcholine-induced relaxation tended to be improved in Gp 3. The amount of eNOS mRNA was increased in Gp 4, and aortic cyclic GMP concentration showed the same tendency. O(2)(-) release tended to be decreased in Gps 2 and 3. The matrix metalloproteinase-1 (MMP-1)-positive area was decreased, and the percentage ratio of cell numbers of smooth muscle cells/macrophages in the plaque was increased in Gp 3. The results demonstrated that sarpogrelate HCl retards the progression of atherosclerosis in rabbits, and that this effect is enhanced by concomitant administration of vitamin E. Although upregulation of eNOS may play a role as one of the underlying mechanisms, our results suggest that an additional mechanism-possibly involving the antiproliferative effects of sarpogrelate HCl on smooth muscle cells and macrophages-may also play an important role.

Topics: Acetylcholine; Animals; Antioxidants; Aorta, Thoracic; Biomarkers; Blood Proteins; Cholesterol, HDL; Coronary Artery Disease; Cyclic GMP; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Endothelium, Vascular; Hypercholesterolemia; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Models, Cardiovascular; Myocardial Contraction; Myocytes, Smooth Muscle; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rabbits; RNA, Messenger; Serotonin Antagonists; Succinates; Treatment Outcome; Vasodilator Agents; Vitamin E

We examined whether oral folate supplementation would rescue a hypercholesterolemia (HC)-related impairment of ischemia-induced angiogenesis.. Folate protects against endothelial dysfunction, but the effect of folate supplementation on angiogenesis is little known.. Sprague-Dawley rats were divided into four groups. Control rats were fed a normal diet (n = 18); HC rats (n = 18) were fed 2% cholesterol diet; and HC + folate (HC+F) rats were fed an HC diet with oral folate (0.003% in water). The left femoral artery and vein were surgically excised, and angiogenesis in the ischemic limb was evaluated. We also examined the effects of Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, on angiogenesis in the HC+F state.. Laser Doppler blood flow (LDBF) analysis showed lower ischemic/normal LDBF ratio in the HC group than in the control group. Angiographic and histologic analyses on day 14 revealed a smaller angiographic score (p < 0.001) and capillary density (p < 0.001) in the HC group than in controls, which were associated with reduced tissue NOx and cyclic guanosine monophosphate (cGMP) levels. The LDBF ratio, angiographic score, and capillary density were significantly restored in the HC+F group (p < 0.01 vs. HC), which were associated with increased serum folate and tissue NOx and cGMP levels. Finally, L-NAME treatment abolished the beneficial action of folate on angiogenesis in the HC state.. Ischemia-induced angiogenesis was inhibited by HC, which was rescued by oral folate supplementation, at least in part, via an NO-dependent manner.

Topics: Administration, Oral; Animals; Cholesterol; Cholesterol, HDL; Cyclic GMP; Dietary Supplements; Disease Models, Animal; Drug Evaluation, Preclinical; Folic Acid; Hindlimb; Homocysteine; Hypercholesterolemia; Ischemia; Male; Neovascularization, Pathologic; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Peripheral Vascular Diseases; Rats; Rats, Sprague-Dawley; Risk Factors; Severity of Illness Index

We sought to determine whether hypercholesterolemia impacts on the NO-stimulated activity of platelet soluble guanylyl cyclase (sGC). We investigated two groups of nine New Zealand white rabbits receiving either a standard (NC) or a cholesterol chow (HC, 0.75%) for 15 weeks. The plasma content of cGMP and the specific activity of sGC in intact platelets were measured by a cGMP-specific radioimmunoassay. In HC, 47.9+/-3.1% of the aortic intimal area was covered with atherosclerotic lesions, and plasma cGMP levels (pmol/ml) were increased from 12.6+/-1.2 to 27.9+/-3.5 (P<.0001). In striking contrast, hypercholesterolemia had no effect on sGC activity stimulated by the NO donor SNAP. At 100 microM SNAP, the specific activities of sGC (pmol/10(9) platelets/min) were 81.8+/-14.5 in NC and 86.2+/-8.1 in HC. Basal sGC activity (pmol/10(9) platelets/min) was also similar in NC (0.21+/-0.04) and HC (0.460+/-0.11, P=.7813). In accordance, washed platelets from both groups showed a similar SNAP-induced inhibition of aggregation. These data suggest that an impaired response of platelets to NO is most likely not involved in platelet hyperreactivity in hypercholesterolemia.

Topics: Animals; Aorta, Abdominal; Aorta, Thoracic; Arteriosclerosis; Blood Platelets; Cyclic GMP; Guanylate Cyclase; Hypercholesterolemia; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Platelet Aggregation; Platelet Count; Rabbits; Receptors, Cytoplasmic and Nuclear; Solubility; Soluble Guanylyl Cyclase; Tunica Intima

Peroxisome proliferator-activated receptor (PPAR) signaling pathways have been reported to exert anti-inflammatory effects and attenuate atherosclerosis formation. However, the mechanisms responsible for their anti-inflammatory and antiatherosclerotic effects remain largely unknown. The present study tested the hypothesis that a PPARgamma agonist may exert significant endothelial protection by antioxidative and antinitrative effects.. Male New Zealand White rabbits were randomized to receive a normal (control) or a high-cholesterol diet and treated with vehicle or rosiglitazone (a PPARgamma agonist) 3 mg x kg(-1) x d(-1) for 5 weeks beginning 3 weeks after the high-cholesterol diet. At the end of 8 weeks of a high-cholesterol diet, the rabbits were killed, and the carotid arteries were isolated. Bioactive nitric oxide was determined functionally (endothelium-dependent vasodilatation) and biochemically (the phosphorylation of vasodilator-stimulated phosphoprotein, or P-VASP). Vascular superoxide production, PPARgamma, gp91phox, and inducible nitric oxide synthase (iNOS) expression, and vascular ONOO- formation were determined. Hypercholesterolemia caused severe endothelial dysfunction and reduced P-VASP, despite a marked increase in iNOS expression and total NOx production. Treatment with rosiglitazone enhanced PPARgamma expression, improved endothelium-dependent vasodilatation, preserved P-VASP, suppressed gp91phox and iNOS expression, reduced superoxide and total NOx production, and inhibited nitrotyrosine formation.. The PPARgamma agonist rosiglitazone exerted a significant vascular protective effect in hypercholesterolemic rabbits, most likely by attenuation of oxidative and nitrative stresses. The endothelial protective effects of PPARgamma agonists may reduce leukocyte accumulation in vascular walls and contribute to their antiatherosclerotic effect.

Topics: Animals; Antioxidants; Blood Vessels; Carotid Arteries; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Hypercholesterolemia; In Vitro Techniques; Lipids; Male; Nitrates; Nitric Oxide; Nitrites; Rabbits; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Signal Transduction; Thiazolidinediones; Transcription Factors; Tyrosine; Vasodilation

Hypercholesterolemia (HC), a pro-oxidant condition, activates nuclear factor-kappa beta (NF-kappa B) and is associated with coronary endothelial dysfunction. The physiological significance of in vivo chronic antioxidant intervention on HC-induced NF-kappa B activation and coronary endothelial function remains unclear.. Four groups of pigs were studied after 12 weeks of normal diet, normal diet with concomitant antioxidant intervention (100 IU/kg of vitamin E and 1 g of vitamin C daily), 2% HC diet, or HC diet+antioxidant supplementation. NF-kappa B activation and the nitric oxide (NO) pathway were investigated by Western blotting and immunohistochemistry, while oxidative stress was evaluated by coronary artery tissue radical scavenger activity and levels of vitamin E and C. Endothelial function was studied in vitro by coronary vasoreactivity to bradykinin and substance P.. HC animals had increased activation of NF-kappa B, decreased endothelial NO synthase expression, and decreased radical scavenger system activity, associated with impaired coronary endothelial function. Antioxidant supplementation in HC normalized NF-kappa B activation and NO bioactivity, and preserves coronary endothelial function.. This study demonstrates for the first time that in vivo chronic interruption of the endogenous oxidative stress cascade reduces HC-induced NF-kappa B activation and normalizes NO bioactivity in association with preservation of coronary endothelial function. This study suggests a role for increased oxidative stress and NF-kappa B activation in early atherosclerosis.

Topics: Animals; Antioxidants; Ascorbic Acid; Cholesterol; Cyclic GMP; Dietary Supplements; Endothelium, Vascular; Female; Hypercholesterolemia; Muscle, Smooth, Vascular; NF-kappa B; Nitric Oxide Synthase; Oxidative Stress; Swine; Vitamin E

The intimal hyperplasia of vein grafts is a major cause of late graft failure and is more pronounced under hyperlipidemia. We previously reported that endothelial cell (ec)-type nitric oxide synthase (NOS) gene transfer inhibited graft intimal hyperplasia under poor runoff conditions. However, little information is available on either ecNOS gene transfer or intimal thickening under hypercholesterolemia.. Using the hemagglutinating virus of Japan liposomes, bovine ecNOS complentary DNA (5000 hemagglutinating activity units/mL) was transfected intraluminally to the right jugular vein, and these veins were then implanted as reversed vein grafts in an end-to-side fashion to the ipsilateral carotid artery.. The cyclic guanosine 3',5'-monophosphate content of the ecNOS vein significantly increased in the grafts at 4 days after gene transfer, but the levels were only 25% greater than those found in the untreated veins. An immunohistochemical analysis at the same time suggested a large loss of medial smooth muscle cells that might have led to a reduction in the exogenous gene expression. The neointima of the ecNOS grafts was significantly reduced 4 weeks after implantation (P <.05), but the effect of ecNOS was limited to about a 30% inhibition. This reduction was associated with a reduced population of proliferating cells and decreased macrophage accumulation in the graft wall.. These results demonstrated that the ecNOS gene transfer suppressed intimal hyperplasia of the vein grafts under hyperlipidemic conditions. However, this effect may be limited because of the smooth muscle cell loss related to the use of an intraluminal delivery methods. These data lead to speculation that the outcome of ecNOS gene transfer could be improved using different methods of gene delivery.

Topics: Animals; Carotid Arteries; Cattle; Cholesterol, Dietary; Cyclic GMP; Gene Transfer Techniques; Genetic Vectors; Hypercholesterolemia; Hyperplasia; Jugular Veins; Kinetics; Liposomes; Macrophages; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rabbits; Sendai virus; Tunica Intima

Hypercholesterolemia (HC) induces alterations in systemic vascular reactivity, which can manifest as an attenuated endothelium-dependent relaxation, partly consequent to an impairment in nitric oxide (NO) activity. To determine whether experimental HC has a similar effect on renal vascular function, renal artery segments obtained from pigs fed a HC (n=5) or normal (n=5) diet were studied in vitro. Endothelium-dependent relaxation was examined using increasing concentrations of acetylcholine (Ach), calcium ionophore A23187, and Ach following pre-incubation with N(G)-monomethyl-L-arginine or L-arginine (L-ARG). The NO-donor diethylamine (DEA) was used to examine smooth muscle relaxation response and cyclic GMP generation in endothelium-denuded vessels. The expression of endothelial NO synthase (eNOS) in the renal arteries was examined using Western blotting. Endothelium-dependent relaxation to Ach was significantly attenuated in the HC group compared to normal (53.3+/-9.1 vs. 98.8+/-3.7%, P<0.005), but normalized after pre-incubation with L-ARG (82.3+/-13.8%, P=0.21). Receptor-independent endothelium-dependent relaxation to A23187 was also significantly blunted in HC (75.2+/-10.5 vs. 115.5+/-4.2%, P<0. 017). Smooth muscle relaxation and cyclic GMP generation in response to DEA were greater in denuded HC vessels, while relaxation of intact vessels to nitroprusside was unaltered. In the HC vessels eNOS was almost undetectable. In conclusion, experimental HC attenuates in vitro endothelium-dependent relaxation of the porcine renal artery, possibly due to low bioavailability of NO. These vascular alterations in HC could play a role in the pathogenesis of renal disease or hypertension, supporting a role for HC as a risk factor for renovascular disease.

Topics: Animals; Arginine; Cholesterol; Cyclic GMP; Diethylamines; Endothelium, Vascular; Hemodynamics; Hypercholesterolemia; In Vitro Techniques; Nitric Oxide; Nitric Oxide Donors; Reference Values; Renal Circulation; Swine; Vasomotor System

We have shown that acute exposure of oxidized low-density lipoprotein (OX-LDL) induces vasoconstriction in renal vessels and reduces glomerular filtration rate (GFR) in an isolated perfused rat kidney model by decreasing the activity of nitric oxide (NO). L-arginine has a protective role against OX-LDl-induced vasoconstriction. Micropuncture studies have demonstrated that short-term diet-induced hypercholesterolaemia is associated with decreased GFR and renal blood flow and increased glomerular capillary pressure. This may be mediated by decreased activity of NO.. Rats were made hypercholesterolaemic by supplementing the standard chow with 4% cholesterol and 1% sodium cholate. A group of rats on hypercholesterolaemic diet also received L-arginine in the drinking water. After 4 and 6 weeks, blood samples and 24-h urine samples were collected for the measurement of biochemical parameters. After 6 weeks, all rats were subjected to isolated perfusion of kidneys at a constant pressure of 100 mmHg. During isolated perfusion, the unused contralateral kidney was taken for morphological studies and for assessing the activity of nitric oxide synthase enzyme by beta-NADPH diaphorase histochemistry.. Rats fed a high-cholesterol diet had LDL levels 3-6 times greater than the rats fed standard chow. Rats that received L-arginine in the drinking water had serum L-arginine levels 5-6 times greater than control rats. At 6 weeks, creatinine clearance was significantly lower in the rats on the high-cholesterol diet compared to the rats on standard chow and rats on high-cholesterol diet plus L-arginine. Twenty-four-hour urinary total nitrate and nitrite excretion in the hypercholesterolaemic rats was 1.5-2 times greater than that of control rats. Twenty-four-hour urinary cGMP excretion was significantly lower in the rats on a high-cholesterol diet, but in the rats on high-cholesterol diet and L-arginine, 24-h urinary cGMP excretion was not significantly different from that of control rats. During isolated perfusion of kidneys, renal perfusate flow was found to be significantly reduced in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation in the drinking water almost completely reversed the effect of a high-fat diet. Inulin clearance was also significantly reduced in kidneys on a high-fat diet in contrast to controls but not in kidneys on high fat-diet and L-arginine. Basal cGMP excretion in urine was significantly lower in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation restored the basal cGMP excretion in these kidneys. NO synthase (NOS) enzyme activity as assessed by NADPH diaphorase activity showed that kidney sections taken from the rats on a high-fat diet showed more intense staining, indicating increased activity compared to the kidney sections taken from the rats on a normal diet.. Though activity of NO is diminished in hypercholesterolaemic rats with impaired renal function, there is a paradoxical increase in NO production and NOS activity. L-arginine reverses the effects of a high-fat diet.

Topics: Animals; Arginine; Cholesterol, Dietary; Cyclic GMP; Hypercholesterolemia; Kidney; Male; NADPH Dehydrogenase; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Rats; Rats, Sprague-Dawley; Reference Values

Hypercholesterolemia impairs systemic vascular reactivity in response to endothelium-dependent vasodilators, which may be mediated partly through increased formation of lipid peroxides. However, it is unclear whether these pathophysiological mechanisms play a role in renal vascular impairment in experimental hypercholesterolemia. Hence, pigs were studied after a 3-mo normal (n = 7) or high cholesterol (HC) (n = 7) diet, HC diet supplemented daily with antioxidant vitamins E (100 IU/kg) and C (1000 mg; HC+vitamins, n = 5), or normal diet supplemented with vitamins (N+vitamins, n = 5). Renal blood flow was measured with electron-beam computed tomography before and during infusion of acetylcholine (Ach). Endothelial function, endothelial and inducible nitric oxide synthase (NOS), and nitrotyrosine immunoreactivity were studied in renal arteries ex vivo. Despite similar cholesterol levels, LDL oxidizability (lag time, malondialdehyde, and relative electrophoretic mobility) was increased in pigs that were fed the HC diet but was significantly decreased in pigs that were fed the HC+vitamins diet. Renal blood flow response to Ach was blunted in pigs that were fed the HC diet but was preserved in pigs that were fed the HC+vitamins diet. Maximal relaxation to Ach was attenuated in pigs that were fed the HC diet compared with those that were fed the normal diet (51.5 +/- 6.4% versus 97.0 +/- 2.9%; P < 0.01) but was preserved in pigs that were fed the HC+vitamins diet (103.1 +/- 3.0%; P = 0.39) and N+vitamins diet (87.7 +/- 3.0%; P = 0.1), as were relaxation responses to calcium ionophore A23187. Vascular smooth-muscle relaxation to diethylamine was enhanced in endothelium-denuded HC vessel but was restored in pigs that were on the HC+vitamins regimen. In HC, immuno-reactivity of endothelial NOS was decreased, that of inducible NOS was increased, and both were preserved in pigs that were fed the HC+vitamins and N+vitamins diets, whereas nitrotyrosine was not detected. The present study demonstrates that antioxidant intervention in experimental HC reduces LDL oxidizability and preserves renal vascular responses to endothelium-dependent vasodilators. Therefore, this beneficial effect potentially can protect the kidney from hypercholesterolemia-induced damage.

Topics: Animals; Antioxidants; Ascorbic Acid; Cholesterol; Cholesterol, Dietary; Cyclic GMP; Endothelium, Vascular; Hemodynamics; Hypercholesterolemia; Immunohistochemistry; In Vitro Techniques; Lipoproteins, LDL; Muscle, Smooth, Vascular; Reference Values; Renal Circulation; Swine; Time Factors; Tomography, X-Ray Computed; Vitamin E

The effects of Western-type fat diet on endothelium-dependent relaxations and vascular structure in carotid arteries from a mouse model of human atherosclerosis are not known. Our objective was to characterize the mechanisms underlying endothelial dysfunction in apoE-deficient mice.. C57BL/6J and apoE-deficient mice were fed for 26 weeks with a lipid-rich Western-type diet. Changes in the intraluminal diameter of pressurized common carotid arteries (ID 450 micrometer) were measured in vitro with a video dimension analyzer. Endothelial NO synthase protein content was evaluated by Western blotting. Intracellular cGMP and cAMP levels were determined by radioimmunoassay.. No morphological changes were observed in the carotid arteries of apoE-deficient mice. However, endothelium-dependent relaxations to acetylcholine (10(-9) to 10(-5) mol/L) were impaired (maximal relaxation 52+/-7% versus 83+/-5% for control mice, P<0.05). Treatment of arteries with NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester inhibited relaxations to acetylcholine to the same extent in apoE-deficient mice as in control mice. Preincubation of carotid arteries with cell-permeable superoxide dismutase mimetic Mn(III) tetra(4-benzoic acid)porphyrin chloride almost normalized NO-mediated relaxations to acetylcholine (75+/-5%, P<0.05). Endothelium-dependent relaxations to calcium ionophore and endothelium-independent relaxations to NO donor diethylammonium(Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate were unchanged in apoE-deficient mice. In addition, no changes in endothelial NO synthase protein expression and cGMP/cAMP levels were found in carotid arteries of apoE-deficient mice.. In carotid arteries of apoE-deficient mice, hypercholesterolemia causes impairment of receptor-mediated activation of eNOS. Increased superoxide anion production in endothelial cells appears to be coupled to activation of cholinergic receptors and is responsible for hypercholesterolemia-induced endothelial dysfunction. The apoE-deficient mouse carotid artery is a valuable new experimental model of endothelial dysfunction.

Topics: Animals; Apolipoproteins E; Carotid Artery, Common; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Free Radical Scavengers; Hypercholesterolemia; Male; Metalloporphyrins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Superoxides; Vasodilation

We examined the implications of iNOS in atherosclerosis progression using the selective inducible NO synthase (iNOS) inhibitor N:-iminoethyl-L-lysine (L-NIL) in hypercholesterolemic rabbits.. Nine rabbits were fed a 0.3% cholesterol diet for 24 weeks (Baseline group); 25 animals were maintained on the diet and treated for 12 extra weeks with L-NIL (5 mg x kg(-1) x d(-1), L-NIL group, n=8), vehicle (Saline group, n=9), or L-arginine (2.25%, L-Arg group, n=8). In abdominal aortas of Saline rabbits, the lesions (53.7+/-5.7%, Baseline) increased to 75.0+/-5.0% (P:<0.05) but remained unaltered in the L-NIL group (63. 4+/-6.6%). Similar results were obtained for the intima/media ratio in thoracic aortas. In coronary arteries, the intima/media ratio was comparable in Baseline (0.68+/-0.18) and Saline (0.96+/-0.19) rabbits but decreased to 0.34+/-0.19 (P:<0.05) in L-NIL rabbits. L-Arginine had beneficial effects only in abdominal aortas. An increased thoracic aorta collagen content was found in Saline and L-Arg but not in L-NIL rabbits. In thoracic aortas of the Saline group, acetylcholine caused modest relaxations that slightly increased by L-arginine but not by L-NIL. Relaxations to nitroglycerin were ameliorated by L-NIL.. This is the first study showing that chronic treatment with an iNOS inhibitor, L-NIL, limits progression of preexisting atherosclerosis in hypercholesterolemic rabbits. Increased intimal collagen accumulation may participate in iNOS-induced atherosclerosis progression.

Topics: Animals; Aorta, Thoracic; Arginine; Blood Cell Count; Cholesterol, Dietary; Collagen; Coronary Artery Disease; Coronary Vessels; Cyclic GMP; Enzyme Inhibitors; Hemodynamics; Hypercholesterolemia; Immunohistochemistry; Lysine; Macrophages; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rabbits; T-Lymphocytes

Endothelium-derived nitric oxide (EDNO) plays an important role in the regulation of angiogenesis, whereas hypercholesterolemia (HC) impairs EDNO release. We examined the hypothesis that HC may inhibit ischemia-induced angiogenesis by inhibition of EDNO in a rat model of unilateral hindlimb ischemia and that oral L-arginine supplementation, a substrate for NO synthase, may prevent HC-related impairment of angiogenesis.. Male Sprague-Dawley rats were fed (A) standard diet (control), (B) 2% high-cholesterol diet (HC group), or (C) high-cholesterol diet with oral L-arginine (2.25% in drinking water) (HC+L-arg group). At 2 weeks of the dietary intervention, unilateral limb ischemia was surgically induced in all animals. Dietary HC groups (B and C) revealed elevated total and LDL cholesterol levels compared with control animals. Laser Doppler blood flow analyses showed significant decreases in the ischemic/normal limb blood flow ratio in the HC group compared with controls (P:<0.05) when followed up until 4 weeks after surgery. Selective angiography and immunohistochemical analyses in the ischemic limb at postoperative day 14 revealed significantly lower angiographic scores (P:<0.01) and capillary densities (P:<0.01) in the HC group than controls, which were associated with decreased tissue contents of NO(x) and cGMP. Oral L-arginine supplementation (HC+L-arg) significantly improved all parameters of the laser Doppler blood perfusion ratio, angiographic scores, and capillary densities (P:<0.01 versus HC group), which were accompanied by significant elevations in serum L-arginine levels and tissue NO(x) and cGMP contents.. Collateral vessel formation and angiogenesis in response to hindlimb ischemia were significantly attenuated in rats with dietary HC. The mechanism may be related to the reduced NO bioactivity in the ischemic tissues. Augmentation of the tissue NO activity by oral L-arginine supplementation restored the impaired angiogenesis in HC.

Topics: Administration, Oral; Animals; Arginine; Body Weight; Cholesterol, Dietary; Collateral Circulation; Cyclic GMP; Endothelium, Vascular; Hindlimb; Hypercholesterolemia; Immunohistochemistry; Ischemia; Laser-Doppler Flowmetry; Lipids; Male; Neovascularization, Physiologic; Nitrates; Nitric Oxide; Nitrites; Rats; Rats, Sprague-Dawley; Regional Blood Flow

To investigate the role of hypercholesterolemia in the regulation of blood pressure.. We compared blood pressure responses to arithmetic stress and hand-grip tests in normotensive patients with hypercholesterolemia n = 15) and a mean (+/- SEM) age of 49 +/- 3 years, and normal cholesterolemic controls (n = 22) aged 48 +/- 1 years. Blood pressure and heart rate were measured throughout the tests. We examined the intracellular Ca2+ concentration in platelets with or without low-density-lipoprotein stimulation (2.9 nmol/l, 10 mg/ml). The plasma nitrite plus nitrate and cyclic GMP were determined before and at the end of each test to evaluate nitric oxide production and activity.. Both tests showed that systolic/diastolic blood pressure was higher in the hypercholesterolemic patients than in the normal controls (stress test: 139 +/- 3/91 +/- 4 versus 127 +/- 2/80 +/- 3 mmHg, P < 0.01/P < 0.05; hand-grip test: 164 +/- 5/106 +/- 5 versus 144 +/- 3/88 +/- 3 mmHg, P < 0.01/P < 0.01). The intracellular Ca2+ concentration in platelets and the increase in response to low-density-lipoprotein stimulation were higher in the hypercholesterolemic patients (without stimulation: 72 +/- 3 versus 64 +/- 3 nmol/l, P < 0.01; with 2.9 nmol/l stimulation: 145 +/- 21 versus 89 +/- 6 nmol/l, P < 0.01). The increase in Ca2+ in response to 2.9 nmol/ml stimulation with low-density lipoprotein was positively related to the increase in mean blood pressure in response to the stress test (r = 0.56, P < 0.002). Nitric oxide production appeared to be increased in the hypercholesterolemic patients (65 +/- 5 versus 51 +/- 4 mmol/l, P < 0.05), and was not affected significantly by the tests. In contrast, cyclic GMP was lower in the patients and was increased significantly in the normal controls by the hand-grip test (P < 0.05). As a result, plasma cyclic GMP was lower in the patients (1.9 +/- 0.2 versus 2.5 +/- 0.1 nmol/l, P < 0.01). The ratio of plasma cyclic GMP to nitric oxide was also lower in the hypercholesterolemic patients at rest (P < 0.05), and at the end of the mental stress (P < 0.02) and hand-grip (P < 0.001) tests.. Patients with hypercholesterolemia showed an exaggerated blood pressure response to both mental stress and exercise, even if resting blood pressure was normal. Increases in the intracellular Ca2+ concentration can contribute to these excessive responses. A disproportionately lower level of cyclic GMP to nitric oxide in plasma may also be involved in these abnormal responses.

Topics: Baroreflex; Blood Platelets; Blood Pressure; Calcium; Cyclic GMP; Heart Rate; Humans; Hypercholesterolemia; Intracellular Fluid; Lipoproteins, LDL; Male; Middle Aged; Nitrates; Nitric Oxide; Nitrites; Pressoreceptors; Stimulation, Chemical; Stress, Mechanical; Stress, Psychological

Shear forces induce platelet aggregation and stimulate the endothelial production of anti-aggregatory factors. Among them, endothelin-3 (ET-3) has been reported to reduce aggregation and to increase platelet cyclic GMP (cGMP) content. Since hypercholesterolemia modifies both platelet aggregability and endothelial function, we compared in 14 hypercholesterolemic and 15 normocholesterolemic subjects the influences of shear forces (240 and 650 s(-1)) on platelet aggregation and cGMP content, and their modulation by ET-3. Spontaneous maximal aggregation occurred earlier and at a greater extent in hypercholesterolemic than in normocholesterolemic subjects (63+/-2 vs 46+/-6% P < 0.01). Pre-treatment with ET-3 abolished the shear-induced facilitation of maximal aggregation in platelets of normocholesterolemic (from 70+/-2 to 52+2% at 240 s(-1) and from 73+/-1 to 59+/-2S at 650s(-1); P < 0.05) and hypercholesterolemic (from 78+/-3 to 64+/-2 at 240 s(-1) and from 78+/-2 to 66+/-3 at 650 s(-1); P < 0.05) subjects. cGMP content did not significantly differ between normocholesterolemic and hypercholesterolemic subjects (6.1+/-0.5 vs 6.9+/-0.7 pmol/10(9) platelets). It was reduced in platelets submitted to shear forces (P < 0.05). This shear-dependent reduction was suppressed by ET-3 pre-treatment. These results demonstrate that shear forces enhance platelet aggregation and diminish their cGMP content. ET-3 reduces the pro-aggregating effects of shear, suggesting a rise in cGMP content as a dynamic associated mechanism.

Topics: Blood Platelets; Cyclic GMP; Endothelin-3; Hemorheology; Humans; Hypercholesterolemia; In Vitro Techniques; Male; Platelet Aggregation; Stress, Mechanical

Field stimulation relaxed the rabbit sphincter of Oddi muscle rings after incubation with atropine (1 microM) and guanethidine (4 microM) with a threefold increase in tissue cyclic cGMP content, a response previously shown to be essentially nitrergic. Preparations from hypercholesterolaemic rabbits (1.5% dietary cholesterol load over 8 weeks increasing serum total cholesterol from pre-diet 1.4+/-0.3 to 22.6+/-3.8 mmol/l) exhibited contractions with no change in cyclic GMP content under the same conditions. The nitrergic relaxation was recaptured with a twofold increase in tissue cyclic GMP content in preparations from hypercholesterolaemic rabbits undergone a treatment with 30 microM/kg farnesol i.v. twice a day over the last 3 days of the dietary period. We conclude that farnesol treatment restores nitrergic relaxation of the sphincter of Oddi in hypercholesterolaemia.

Topics: Animals; Cyclic GMP; Farnesol; Hypercholesterolemia; Isometric Contraction; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Rabbits; Sphincter of Oddi

The negative charges of dextran sulfate (DS) used for low-density lipoprotein (LDL) apheresis activate the intrinsic coagulation pathway, accompanied by the production of bradykinin. This study was undertaken to see whether cyclic nucleotide plasma levels are affected by DS LDL apheresis. Previously, we showed the rise in plasma levels of prostaglandins and nitric oxide derivatives accompanied by the rise in bradykinin levels. The physiologic effects of prostaglandins and nitric oxide become manifest through the intracellular signal of cyclic nucleotides. The plasma levels of the cyclic nucleotides (cyclic adenosine monophosphate [cAMP] and cyclic guanosine monophosphate [cGMP]) were examined when either of 2 anticoagulants, heparin or nafamostat mesilate (NM), was used during DS LDL apheresis. The plasma levels of cAMP during LDL apheresis using heparin were 9.2 +/- 0.3 (mean +/- SE) 12.4 +/- 0.6, 12.0 +/- 0.5, and 12.1 +/- 0.3 pmol/ml, respectively, at the 0, 1,000, 2,000, and 3,000 ml stages. The rise in cAMP levels was suppressed during apheresis using NM. There were no significant increases in cGMP during apheresis with heparin or with NM. There were significant negative correlations between changes in cAMP and those in the blood pressure. These findings suggest that bradykinin generated during apheresis exerts some physiologic effects via activation of the adenylate cyclase dependent pathway.

Topics: Adrenomedullin; Anticoagulants; Benzamidines; Blood Component Removal; Bradykinin; Cyclic AMP; Cyclic GMP; Dextran Sulfate; Guanidines; Heparin; Humans; Hypercholesterolemia; Lipoproteins, LDL; Male; Middle Aged; Nitric Oxide; Nucleotides, Cyclic; Peptides; Prostaglandins

The response to nitric oxide of intracellular free Ca2+ levels, measured by fura 2 fluorimetry, and cyclic GMP, measured by RIA, was evaluated on smooth muscle cells of the thoracic aorta in primary culture from normal and cholesterol-fed rabbits. Relaxation to acetylcholine and nitric oxide was also determined in isolated rings of aorta. After 10 weeks of high-cholesterol diet, the intact aorta relaxed less to both acetylcholine and nitric oxide. In cultured cells from hypercholesterolemic rabbits, intracellular Ca2+ oscillated, and the mean Ca2+ levels were approximately twofold greater than in normal aortic cells. Nitric oxide failed to affect basal Ca2+ in either cell type. The peak and sustained rise in intracellular Ca2+ induced by angiotensin II (10(-7) mol/L) were similar in the two cell types. However, nitric oxide (10(-10) to 10(-6) mol/L) decreased the sustained Ca2+ levels to a significantly smaller extent in cells from cholesterol-fed rabbits. In addition, in cells from hypercholesterolemic rabbits, nitric oxide added before angiotensin II inhibited to a smaller degree the transient increase in intracellular free Ca2+ caused by angiotensin II in the nominal absence of extracellular Ca2+, as well as the increase in Ca2+ associated with the addition of extracellular Ca2+. Measurements of fura 2 quenching caused by Mn2+ influx confirmed that nitric oxide inhibited the entry of extracellular divalent cations significantly less in cells from hypercholesterolemic rabbits. Basal levels of cyclic GMP were significantly less than normal, and nitric oxide increased levels of cyclic GMP to a significantly smaller degree in cells from cholesterol-fed rabbits. These data indicate a substantial resistance to nitric oxide action in aortic smooth muscle cells of cholesterol-fed rabbits. This observation is consistent with the notion that resistance of smooth muscle cells to nitric oxide contributes to abnormal endothelium-dependent vasodilation during hypercholesterolemia and can play a role in the pathogenesis of atherosclerosis.

Topics: Animals; Aorta; Calcium; Cyclic GMP; Hypercholesterolemia; Intracellular Membranes; Male; Muscle, Smooth, Vascular; Nitric Oxide; Rabbits; Vasodilation

We investigated whether L-arginine induces regression of preexisting atheromatous lesions and reversal of endothelial dysfunction in hypercholesterolemic rabbits, whether similar effects can be obtained by cholesterol-lowering therapy with lovastatin, and which mechanism leads to these effects.. Rabbits were fed 1% cholesterol for 4 weeks and 0.5% cholesterol for an additional 12 weeks. Two groups of cholesterol-fed rabbits were treated with L-arginine (2.0% in drinking water) or lovastatin (10 mg/d) during weeks 5 through 16. Systemic nitric oxide (NO) formation was assessed as the urinary excretion rates of nitrate and cGMP in weekly intervals. Cholesterol feeding progressively reduced urinary nitrate excretion to approximately 40% of baseline (P<.05) and increased plasma concentrations of asymmetrical dimethylarginine (ADMA), an endogenous NO synthesis inhibitor. Dietary L-arginine reversed the reduction in plasma L-arginine/ADMA ratio and partly restored urinary excretion of nitrate and cGMP (each P<.05 vs cholesterol) but did not change plasma cholesterol levels. L-Arginine completely blocked the progression of carotid intimal plaques, reduced aortic intimal thickening, and preserved endothelium-dependent vasodilator function. Lovastatin treatment reduced plasma cholesterol by 32% but did not improve urinary nitrate or cGMP excretion or endothelium-dependent vasodilation. Lovastatin had a weaker inhibitory effect on carotid plaque formation and aortic intimal thickening than L-arginine. L-Arginine inhibited but lovastatin potentiated superoxide radical generation in the atherosclerotic vascular wall.. Dietary L-arginine improves NO-dependent vasodilator function in cholesterol-fed rabbits and completely blocks the progression of plaques via restoration of NO synthase substrate availability and reduction of vascular oxidative stress. Lovastatin treatment has a weaker inhibitory effect on the progression of atherosclerosis and no effect on vascular NO elaboration, which may be due to its stimulatory effect on vascular superoxide radical generation.

Topics: Animals; Anticholesteremic Agents; Arginine; Arteriosclerosis; Cholesterol; Cholesterol, Dietary; Cyclic GMP; Diet, Atherogenic; Endothelium, Vascular; Hypercholesterolemia; Lovastatin; Male; Nitrates; Nitric Oxide; Rabbits; Superoxides

To determine if endogenous local levels of nitric oxide (NO) modulate atherogenesis, we studied the effect of inhibiting NO with NG-nitro-L-arginine methyl ester (L-NAME) on early neointima formation in cholesterol-fed rabbits. Male rabbits were fed for 5 weeks with a 0.5% cholesterol diet alone or treated in addition during the last 4 weeks with L-NAME (12 mg/kg per day SC) via osmotic minipump. Endothelial cell function was assessed in isolated aortic rings by vascular reactivity and levels of cyclic GMP. In L-NAME-treated rabbits there was inhibition of endothelium-dependent relaxations to acetylcholine and the calcium ionophore A23187 as well as impaired cyclic GMP accumulation in response to acetylcholine. Neointima formation in the ascending thoracic aorta was assessed by determining media and intima cross-sectional areas with computerized image analysis. Compared with rabbits that consumed the cholesterol diet alone, L-NAME-treated rabbits had significant increases in lesion area (0.29 +/- 0.04 versus 0.15 +/- 0.03 mm2) and in lesion/media ratio (0.06 +/- 0.01 versus 0.03 +/- 0.01). Plasma levels of cholesterol and fluorescent lipid peroxide products were unchanged, suggesting no difference in cholesterol metabolism or oxidation. Because arterial blood pressure was not altered by L-NAME treatment, the increased atherogenesis could not be attributed to an increase in blood pressure. These results indicated that local inhibition of NO accelerates early neointima formation possibly because of modulating monocyte recruitment or foam cell lipid accumulation.

Topics: Amino Acid Oxidoreductases; Animals; Aorta; Arginine; Arteriosclerosis; Cyclic GMP; Endothelium, Vascular; Hypercholesterolemia; Lipid Peroxidation; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Rabbits; Vasodilation

The effect of hypercholesterolemia for 10 wk on endothelium-dependent relaxations to acetylcholine was studied in isolated rings of rabbit carotid artery and abdominal aorta contracted with phenylephrine or elevated potassium. In these arteries obtained from hypercholesterolemic rabbits, endothelium-dependent relaxations to acetylcholine were not significantly different from those of normal rabbits. In normal and hypercholesterolemic arteries, partial relaxation persisted in the presence of NG-nitro-L-arginine methyl ester (L-NAME), which blocked acetylcholine-induced increases in arterial guanosine 3',5'-cyclic monophosphate (cGMP). Combined treatment with L-NAME and the calcium-dependent potassium-channel inhibitor, charybdotoxin, blocked relaxations in both groups, suggesting that L-NAME-resistant relaxations are mediated by an endothelium-derived hyperpolarizing factor. Charybdotoxin alone or depolarizing potassium had no significant effect on normal carotid artery or normal and hypercholesterolemic abdominal aorta but significantly inhibited relaxations of the carotid artery from cholesterol-fed rabbits. The enhanced role of calcium-dependent potassium channels and the hyperpolarizing factor in relaxation of the hypercholesterolemic carotid artery suggested by these results was likely related to the fact that acetylcholine failed to stimulate cGMP only in that artery. These data suggest that endothelium-dependent relaxation in these rabbit arteries is mediated by nitric oxide-cGMP-dependent and -independent mechanisms. In hypercholesterolemia, the contribution of nitric oxide-cGMP in the carotid artery is reduced, but a hyperpolarizing factor and calcium-dependent potassium channels maintain normal acetylcholine-induced relaxation.

Topics: Acetylcholine; Analysis of Variance; Animals; Arginine; Carotid Arteries; Charybdotoxin; Cholesterol, Dietary; Cyclic GMP; Dose-Response Relationship, Drug; Hypercholesterolemia; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Potassium; Potassium Channels; Rabbits; Reference Values; Scorpion Venoms

Platelets are capable of producing nitric oxide (NO) through the L-arginine-NO synthase pathway. Acute exposure to supraphysiological concentrations of L-arginine in vitro increases the production of NO by platelets and is associated with an increase in platelet cyclic GMP (cGMP) levels and a reduction in platelet aggregation. The purpose of this study was to determine if chronic oral administration of L-arginine decreases platelet aggregation in hypercholesterolemic animals and to determine if this effect is mediated by the metabolism of L-arginine to NO. Male New Zealand White rabbits were fed normal chow (Con), a 1% cholesterol diet (Chol), or a 1% cholesterol diet supplemented with a sixfold enrichment of dietary L-arginine (Arg) or L-methionine (Met). After 10 weeks, cholesterol levels were equally increased in Chol and Arg animals, whereas plasma arginine levels were doubled in the Arg group. There was no difference in maximum aggregation initiated by ADP (100 mumol/L) between washed platelets from Con, Met, and Chol animals, but aggregation of platelets from Arg animals was significantly decreased (P < .05). In aggregating platelets from Arg animals, cGMP levels were significantly higher than the other groups (P < .05). When platelets were incubated ex vivo with the NO synthase inhibitor NG-monomethyl-L-arginine, the effects of dietary L-arginine were reversed. Chronic dietary supplementation of L-arginine decreases platelet aggregation in hypercholesterolemic rabbits. This effect appears to be due to the metabolism of L-arginine to NO.

Topics: Adenosine Diphosphate; Animals; Arginine; Blood Platelets; Calcium; Cholesterol; Cholesterol, Dietary; Cyclic GMP; Hypercholesterolemia; Intracellular Membranes; Male; Platelet Aggregation; Rabbits