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Although endothelium-dependent vasodilatation has been used as a marker of endothelial dysfunction (ED), there have been no reliable plasma markers for ED. Oxidative stress, which is a major determinant of ED, oxidizes tetrahydrobiopterin (BH4), an essential cofactor of endothelial type nitric oxide synthase (eNOS), and resulted in the relative deficiency of BH4.In 163 patients with cardiovascular disorders, the plasma levels of BH4 and 7, 8-dihydrobiopterin (BH2) by high performance liquid chromatography were measured and compared with the flow-mediated (FMD) vasodilatory response of the brachial artery, which was measured by ultrasonography. The effects of atorvastatin on plasma pteridine levels and FMD were examined in patients with multiple coronary risk factors. There was a positive relationship between FMD and plasma BH4 levels and a negative relationship between FMD and plasma BH2 levels. Subsequently, a strong positive relationship between FMD and the BH4/BH2 ratio (r=0.585, P0.0001) was found. Although we did not find any significant relationship between pteridine levels and individual traditional risk factors, the BH4/BH2 ratio in patients with more than 2 risk factors showed significant reductions compared with that in those without risk factors. Statin treatment improved FMD in association with an increase in the plasma BH4/BH2 ratio.Plasma pteridine levels were associated with endothelial dysfunction in cardiovascular disorders.

Objective— Diabetes mellitus is associated with increased oxidative stress, which induces oxidation of tetrahydrobiopterin (BH4) in vessel wall. Without enough BH4, eNOS is uncoupled to L-arginine and produces superoxide rather than NO. We examined the role of uncoupled eNOS in vascular remodeling in diabetes. Methods and Results— Diabetes mellitus was produced by streptozotocin in C57BL/6J mice. Under stable hyperglycemia, the common carotid artery was ligated, and neointimal formation was examined 4 weeks later. In diabetic mice, the neointimal area was dramatically augmented. This augmentation was associated with increased aortic superoxide formation, reduced aortic BH4/dihydrobiopterin (BH2) ratio, and decreased plasma nitrite and nitrate (NOx) levels compared with nondiabetic mice. Chronic BH4 treatment (10 mg/kg/d) reduced the neointimal area in association with suppressed superoxide production and inflammatory changes in vessels. BH4/BH2 ratio in vessel wall was preserved, and plasma NOx levels increased. Furthermore, in the presence of diabetes, overexpression of bovine eNOS resulted in augmentation of neointimal area, accompanied by increased superoxide production in the endothelium. Conclusions— In diabetes, increased oxidative stress by uncoupled NOSs, particularly eNOS, causes augmentation of vascular remodeling. These findings indicate restoration of eNOS coupling has an atheroprotective benefit in diabetes.

Background: Reliable non-invasive imaging modalities to characterize atherosclerotic plaque components are clinically desirable for detecting unstable coronary plaques, which cause acute coronary syndrome or sudden death. Although recent clinical developments in computed tomography (CT) have enabled the visualization of luminal narrowing and calcified plaques in coronary arteries, the evaluation of non-calcified plaque components remains difficult. Phase-contrast X-ray CT imaging has great potential to reveal the structures inside soft tissues because its sensitivity to light elements is almost 1000 times greater than that of absorption-contrast clinical X-ray imaging. We for the first time examined mouse atherosclerotic plaques using phase-contrast X-ray CT and found promising results. Methods and Results: Ex vivo phase-contrast X-ray CT was performed using a synchrotron radiation source (SPring-8, Japan) to investigate atherosclerotic plaque components in mice. Samples were also histologically analyzed. Phase-contrast X-ray CT at a spatial resolution of 10 –20 μm revealed atherosclerotic plaque components, and thin fibrous caps could be easily detected. The specific mass densities of these components were estimated using dδ (differences in the refractive indexes relative to water). While lipid-rich areas showed low dδ (0.79 ± 0.13 × 10 −8 ) and mass density (1.011 ± 0.001 g/ml), the smooth muscle- and collagen-rich areas showed high dδ (4.18 ± 0.10 × 10 −8 and 5.93 ± 0.13 × 10 −8 , respectively) and mass density (1.057 ± 0.001 g/ml and 1.08 ± 0.002 g/ml, respectively). It was rather easy to evaluate or differentiate the atherosclerotic plaque components using this novel phase-contrast X-ray CT imaging. Moreover, the three-dimensional assessment of plaques was possible, and it enabled the imaging of their anatomical information. Conclusions: Phase-contrast X-ray CT can estimate the tissue-mass density of atherosclerotic plaques and distinguish the lipid-rich areas from the collagen-rich areas. This is a promising non-invasive technique for the investigation of plaque components and detection of unstable coronary plaques.

Maternal hypercholesterolemia during pregnancy increases offspring susceptibility to atherosclerosis by an oxidation-dependent mechanism. The present studies investigated whether maternal immunization with oxidized LDL (OxLDL) before pregnancy protects the fetus from atherogenic in utero programming by maternal hypercholesterolemia. Maternal immunization of NZW rabbits and LDL receptor–deficient mice indeed reduced atherosclerosis in adult offspring by up to 56%, but the protective effect could not be attributed to a reduction of fetal exposure to hypercholesterolemia alone, and even nonspecific immune stimulation with adjuvant only provided some protection. Unexpectedly, offspring of immunized mothers developed increased IgM antibodies to selective OxLDL epitopes and increased IgM-LDL immune complexes, compared with offspring of nonimmunized controls. Even naïve offspring of OxLDL-immunized mothers never exposed to postnatal hypercholesterolemia responded to a one-time OxLDL and KLH challenge with greater OxLDL-specific IgM responses, increased OxLDL-specific IgM-secreting B cells, and more IgM-LDL immune complexes. In contrast, maternal immunization with KLH, a T cell–dependent nonmammalian antigen, did not influence postnatal immune responses. Effects of maternal OxLDL-immunization on offspring B cells and selective antibodies were independent of transplacental passage of maternal immunoglobulins. Results show that maternal immunization with antigens prevalent in atherosclerotic lesions reduces atherogenesis in their offspring by mechanisms that include, but are not limited to, reduced fetal exposure to maternal hypercholesterolemia and lipid peroxidation. More importantly, they demonstrate in principle that maternal adaptive immunity to selective antigens influences postnatal B cell and antibody responses in offspring, and that modulation of in utero immune programming may influence immune-modulated diseases later in life.

Background: Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is thought to effect an anti-inflammatory response, but its mechanism is still unknown. Methods: eNOS transgenic (eNOS-TG) mice and their littermate controls (C57/BL6) were used to clarify the role of NO derived from eNOS. Bleomycin hydrochloride (1 U/body/day) or PBS was injected intraperitoneally. Results: Subpleural fibrotic changes and hydroxyproline content in the eNOS-TG mice were significantly reduced compared with those of the wild-type (WT) mice by day 56. Administration of Nω-nitro-l-arginine methyl ester, a potent inhibitor of NO synthase, worsened the fibrotic response in bleomycin-treated eNOS-TG mice. Gelatinolytic activity in lung homogenates, corresponding to metalloproteinase-9 (MMP-9), was significantly increased in bleomycin-injured WT mice on day 14. In contrast, the level of tissue inhibitor of metalloproteinases-1 (TIMP-1), an endogenous MMP-9 inhibitor, was increased in the bleomycin-treated eNOS-TG mice compared with WT. Immunohistochemical analysis demonstrated that MMP-9 and TIMP-1 were strongly expressed in inflammatory cells, including subpleural fibrotic lesions. Conclusion: These data suggested that eNOS overexpression attenuates bleomycin-induced lung injury by ameliorating the MMP-9/TIMP-1 balance.

In vitro and in vivo experimental models have demonstrated that vascular endothelial function is significantly impaired as a result of oxidative stress, mediated by the generation of oxygen-derived free radicals in response to chronic or acute inflammation. In particular, super-oxide () at specific concentrations leads to the impairment of nitric oxide (NO) bioactivity, and it is known that NO plays a fundamental role in the maintenance of vascular homeostasis. The relationship between reactive oxygen species (ROS) and NO release in thrombosis-related endothelial damage in the peripheral microvasculature remains unclear, however. The purpose of the present study was to investigate the effect of the free-radical scavenger, edaravone, on NO synthesis and thrombotic potential in arterioles after exposure to laser irradiation. Highly sensitive electrochemical NO microsensors were positioned in femoral arterioles of mice, and the kinetics of NO release were recorded in response to standardized laser irradiation in vivo. In addition, images of NO release from damaged vascular cells were investigated in a similar rat model using the NO-sensitive dye 4,5-diaminofluorescein diacetate (DAF-2DA). Thrombogenesis was assessed in carotid arterioles by continuous video microscopy using image analysis software. Laser irradiation led to NO release from perturbed endothelial cells and from platelet-rich thrombi. Edaravone had no significant effect on NO release in non-laser treated, intact endothelium compared with placebo. In contrast, edaravone demonstrated a dose-dependent effect on NO release and thrombogenicity. At a concentration of 10.5 mg/kg per h, edaravone promoted a 5-fold increase in NO and a reduction in platelet-rich thrombus volume to 58% of the placebo values. Our data provide direct evidence to confirm that acute endothelial damage in peripheral microvessels initially induces NO release and that the free-radical scavenger, edaravone, augments NO synthesis leading to suppression of platelet thrombus formation.

Although mechanical ventilation (MV) is an important supportive strategy for patients with acute respiratory distress syndrome, MV itself can cause a type of acute lung damage termed ventilator-induced lung injury (VILI). Because nitric oxide (NO) has been reported to play roles in the pathogenesis of acute lung injury, the present study explores the effects on VILI of NO derived from chronically overexpressed endothelial nitric oxide synthase (eNOS). Anesthetized eNOS-transgenic (Tg) and wild-type (WT) C57BL/6 mice were ventilated at high or low tidal volume (Vt; 20 or 7 ml/kg, respectively) for 4 h. After MV, lung damage, including neutrophil infiltration, water leakage, and cytokine concentration in bronchoalveolar lavage fluid (BALF) and plasma, was evaluated. Some mice were given Nω-nitro-l-arginine methyl ester (l-NAME), a potent NOS inhibitor, via drinking water (1 mg/ml) for 1 wk before MV. Histological analysis revealed that high Vt ventilation caused severe VILI, whereas low Vt ventilation caused minimal VILI. Under high Vt conditions, neutrophil infiltration and lung water content were significantly attenuated in eNOS-Tg mice compared with WT animals. The concentrations of macrophage inflammatory protein-2 in BALF and plasma, as well as plasma tumor necrosis factor-α and monocyte chemoattractant protein-1, also were decreased in eNOS-Tg mice. l-NAME abrogated the beneficial effect of eNOS overexpression. In conclusion, chronic eNOS overexpression may protect the lung from VILI by inhibiting the production of inflammatory chemokines and cytokines that are associated with neutrophil infiltration into the air space.

Angiotensin II is involved in the process of atherosclerosis and stimulates superoxide production from cardiovascular cells. We examined the effect of telmisartan, an angiotensin II type 1 receptor blocker, on atherosclerosis. We chronically treated apolipoprotein E-deficient mice with two different doses of telmisartan dissolved in drinking water (0.3 and 3 mg/kg) starting from 4 weeks of age for 12 weeks. Lipid contents were not different in both telmisartan-treated groups compared with control group. Systolic blood pressure was significantly reduced with 3 mg/kg, but unchanged with 0.3 mg/kg. The total atherosclerotic lesion size at the aortic sinus was reduced with 0.3 mg/kg compared with control, and additional reduction was proved with 3 mg/kg. The fibrotic change was not different among three groups, but MOMA-2-, malondialdehyde-, 4-hydroxy-2-nonenal-immunostained areas were reduced by telmisartan. As the mechanism, we revealed that both doses of telmisartan markedly reduced superoxide production from in situ vessels assessed by lucigenin-enhanced chemiluminescence and dihydroethidium staining. And NAD(P)H dependent oxidase activity in vessels was reduced by telmisartan. Further, 8-iso-prostaglandin F2alpha level, a systemic oxidative stress marker, obtained from urine and plasma samples were significantly reduced by telmisartan. Telmisartan reduced atherosclerosis in apolipoprotein E-deficient mice at least partly via the suppression of oxidative stress.

Endothelial lipase (EL) expression correlates inversely with circulating high density lipoprotein (HDL) cholesterol levels in genetic mouse models, and human genetic variation in this locus has been linked to differences in HDL cholesterol levels. These data suggest a role for EL in the development of atherosclerotic vascular disease. To investigate this possibility, LIPG-null alleles were bred onto the apoE knockout background, and the homozygous double knockout animals were characterized. Both apoE knockout and double knockout mice had low HDL cholesterol levels when compared with wild-type mice, but the HDL cholesterol levels of the double knockout mice were higher than those of apoE knockout mice. Atherogenic very low density lipoprotein and intermediate density lipoprotein/low density lipoprotein cholesterol levels of the double knockout mice were also greater than those of the apoE knockout animals. Despite this lipid profile, there was a significant approximately 70% decrease in atherosclerotic disease area in double knockout mice on a regular diet. Immunohistochemistry and protein blot studies revealed increased EL expression in the atherosclerotic aortas of the apoE knockout animals. An observed decrease in macrophage content in vessels lacking EL correlated with ex vivo vascular monocyte adhesion assays, suggesting that this protein can modulate monocyte adhesion and infiltration into diseased tissues. These data suggest that EL may have indirect atherogenic actions in vivo through its effect on circulating HDL cholesterol and direct atherogenic actions through vascular wall processes such as monocyte recruitment and cholesterol uptake.

Background Although incidence of arterial thrombotic events increases with advancing age, the mechanism of such increased risk is poorly understood. There is also a gender difference; coronary heart disease is more common in men than women. The aim of the present study was to investigate the possible contribution from platelet reactivity and spontaneous thrombolysis, determinants of arterial thrombosis, to the increased risk of thromboembolism in aging, but otherwise healthy subjects. Method One hundred and forty-five normal subjects (61 men, 82 women) of age between 19 and 77 years were grouped into groups of young ( 51 years), according to their gender and body mass index (BMI). A new in vitro test [Gorog Thrombosis Test (GTT)] was used to measure in sequence both platelet reactivity [occlusion time (OT)] and spontaneous thrombolysis [lysis time (LT)] from one non-anticoagulated blood sample. Result OT in all women was 315±9.2 s (mean±S.E.M.), in all men OT was 300±10.6 s. The mean LT in all women was 2557±201.5 s, and in all men LT was 2493±198.6 s. Advanced age did not enhance platelet reactivity (OT), but increased BMI did (P=0.039). Spontaneous thrombolysis (LT) was impaired in older men but not in women (difference between young vs. middle age: P=0.019; young vs. older: P=0.0002). Conclusion Our findings suggest that in men, spontaneous thrombolytic activity is reduced with age, and this may explain the increased frequency and severity of thromboembolic events. Interestingly, in women, spontaneous thrombolytic activity did not change after menopause.

Atherosclerosis has a close relationship to inflammation, particularly T helper type 1 lymphocyte (Th1) response. Interleukin-10 (IL-10), is thought to suppress Th1 response. To target therapeutic strategy for atherosclerosis, we tested whether IL-10 gene transfer suppresses atherosclerosis in apolipoprotein E-knockout (apoE-KO) mice. Four-week-old apoE-KO mice were divided into two groups and either murine IL-10 cDNA plasmid or empty control vector was transferred to the femoral muscle with the use of Hemagglutinating virus of Japan (HVJ)-liposome. At 1 week after transfection, high cholesterol diet was started and continued for 8 weeks. After euthanasia, histological studies of atherosclerotic lesions and quantitative RT-PCR for Th1 cytokines (IL-12 and IFN-gamma) in spleens were performed. IL-10 cDNA gene transfer to the muscle increased plasma IL-10 levels and depressed expression of Th1 cytokines without changing plasma cholesterol levels. IL-10 gene transfer significantly reduced the atherosclerotic plaque area and the macrophage infiltrated area. IL-12 and IFN-gamma mRNA expressions in spleens and plasma IFN-gamma levels were decreased by IL-10 gene transfer. Therefore, IL-10 gene transfer changed the Th1 response and suppressed atherosclerotic lesion formation in apoE-KO mice. IL-10 could be a new target as a therapeutic tool for the treatment of atherosclerosis.

The influence of dietary bacillus natto productive protein (BNPP) on endogenous thrombolysis was investigated in the rat. Animals were given a standard feed for 14 weeks, to which 0.2 or 1% BNPP was added. Thrombolysis was evaluated using an He-Ne laser-induced thrombosis model in mesenteric microvessels. Changes in thrombus volume, reflecting thrombolysis, decreased to 82% of the initial value in the control group. In contrast, the thrombus volume decreased to 67% in the animals fed 0.2% BNPP, and decreased to 51% in the group given 1% BNPP. The extent of thrombolysis in the 1% BNPP group was equivalent to that seen in animals treated with a bolus intravenous infusion of 0.2 mg/kg tissue plasminogen activator. The results demonstrated that the dietary administration of BNPP enhanced endogenous thrombolysis in a dose-dependent manner. Argatroban (2 mg/kg/h) enhanced endogenous fibrinolysis only in control animals, but not in the BNPP groups. The results support the suggestion that dietary supplementation with BNPP may provide a simple means to promote fibrinolysis not only in the treatment of thromboembolism but also in the prevention of venous occlusion.

Background Recent studies suggest that 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) exert their protective effects against cardiovascular diseases independently of their cholesterol-decreasing effects. Objective To clarify the effect of a statin on hypertensive nephrosclerosis. Methods We treated stroke-prone spontaneously hypertensive rats (spSHRs) chronically, starting at the age of 4 weeks, with cerivastatin (2 mg/kg per day by gavage) or vehicle. Physiological parameters, plasma chemistry and urine protein excretion were analysed. At 14 weeks of age, the rats had their kidneys removed for use in assays. Results Compared with vehicle treatment, statin treatment reduced proteinuria and renal injury independently of blood pressure and cholesterol concentrations in spSHRs. Although expression of adhesion molecules and infiltration of inflammatory cells were not different whether or not cerivastatin treatment was used, renal fibrosis was significantly reduced in statin-treated spSHRs. We also found that expression of transforming growth factor-β1 in kidneys was significantly inhibited in statin-treated spSHRs. Conclusion Cerivastatin prevents or retards hypertension-induced renal injury via inhibition of renal fibrosis and proteinuria. These results show the potential of statins as protective tools against proteinuric renal diseases, independent of their cholesterol-decreasing effects.

Genetically modified hyperlipidemic mice provide important information on the pathogenesis of atherosclerosis, but most experimental designs are limited to in vitro or ex vivo examinations. The present study was designed to detect atherosclerotic lesions in situ in apolipoprotein E-knockout (apoE-KO) mice using a newly developed angiography system, synchrotron radiation (SR) microangiography, which uses monochromatic SR as an X-ray source and a high definition camera or video system as a detector. Digital microangiography with 7 mm pixel sizes was carried out and atherosclerotic lesion in small arteries less than 500 μm in diameter were detected. Moreover, the coronary artery stenotic lesion of an apoE-KO mouse was detected in situ with the angiography system. The new SR microangiography system is a powerful tool for investigating atherosclerotic lesions in situ in genetically engineered mice and will promote the basic study of atherosclerotic disease. (Circ J 2002; 66: 1057 - 1059)

Monocyte chemoattractant protein-1 (MCP-1), which binds to C-C chemokine receptor 2, has been implicated as the primary source of monocyte chemoattractant function in the early stages of atherosclerosis. Recently, propagermanium, a drug used clinically for the treatment of chronic hepatitis in Japan, has been shown to inhibit C-C chemokine receptor 2 function and suppress monocyte/macrophage infiltration in vitro and in vivo. Given the importance of monocyte infiltration in atherogenesis, the inhibition of it by propagermanium might prevent atherosclerosis. Apolipoprotein E knockout (apoE-KO) mice were fed an atherogenic high cholesterol diet with or without 0.005% propagermanium for 8 or 12 weeks. Although the plasma lipid levels were unchanged by the drug treatment, atherosclerotic lesion area in the aortic root was reduced by 50% in the drug-treated apoE-KO mice compared with the nontreated apoE-KO mice after 8 weeks of cholesterol feeding (0.62±0.12 versus 1.27±0.07 mm 2 , respectively; P 2 [drug-treated group] versus 0.67±0.07 mm 2 [control group]; P

Genetically engineered mice provide enormous potential to study the pathogenesis and treatment of atherosclerosis. However, most experimental designs to investigate mouse coronary atherosclerosis are limited to in vitro and ex vivo examinations. Technological advances in microangiography have facilitated the detailed study of microvessels ex vivo and in vivo; the development of synchrotron radiation (SR) microangiography has enabled us to study mouse coronary arteries in moving hearts. For high-resolution, real-time imaging (10×5 mm, 30 frames/second), a monochromatized x-ray obtained from the …

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a central role in regulation of vascular tone and reactivity. The purpose of this study is to clarify the basal tone and microvascular reactivity in eNOS-overexpressing transgenic (Tg) mice in vivo with a microangiography system using monochromatic synchrotron radiation (SR). The mouse femoral artery was cannulated, nonionic contrast media was injected, and microangiography was performed in hindlimbs of mice. Serial images of the small blood vessels (diameter200 microm) were recorded by the SR microangiography system. At basal conditions, the diameter of tibial arteries in eNOS-Tg mice was larger than that of wild-type mice (179 +/- 8 versus 132 +/- 8 microm; P0.01). l-NAME treatment decreased the vessel diameter and canceled the difference in vessel diameters between two genotypes. Acetylcholine- and sodium nitroprusside-induced relaxations of small vessels were significantly reduced in Tg mice compared with wild-type mice (35.0 +/- 9.4 versus 61.6 +/- 6.7%, 85.0 +/- 10.2 versus 97.3 +/- 6.7% of the maximum relaxation, respectively). Our data provide the evidence that overproduced NO from endothelium reduces vascular tone and plays a pivotal role in regulation of vascular tone in small vessels. Furthermore, the reduced NO-mediated relaxation in small vessels of eNOS-Tg mice is demonstrated for the first time in vivo. SR microangiography allows us to evaluate the reactivity in small-sized vessels and appears to be a powerful tool for assessing the microvascular circulation in vivo.

Recent studies suggest that several calcium channel blockers exert their protective effects against vascular disorders by increasing nitric oxide (NO) production from the endothelium. The purpose of this study was to clarify the effects of a long-lasting calcium channel blocker, benidipine, on vascular remodeling.The left common carotid arteries of mice were completely ligated just proximal to the carotid bifurcation. Treatment with benidipine (3 mg/kg per day) or vehicle was started 1 week before the carotid ligation, and continued throughout the experiments. Four weeks after the carotid ligation, these mice were killed and vascular remodeling was analyzed. Moreover, NO production and endothelial NO synthase (eNOS) expression were assessed.At 4 weeks after ligation, the neointimal area in the vehicle-treated mice was 39,400 +/- 4,900 microm2 (n = 8), whereas that in the drug-treated mice was reduced to 18,300 +/- 3,800 microm2 (n = 10). Consequently, the luminal area was 35% larger in the drug-treated mice. Benidipine increased the basal as well as agonist-induced NO production from the endothelium, detected by Griess method or NOx analyzer. Endothelial NOS expression in vessels of the drug-treated mice was increased compared with that of the vehicle-treated mice.Our data provide evidence that benidipine increases NO production via increment of eNOS protein in vessels and prevents intimal thickening in mice. These results show the possibility of benidipine as a protective tool against vascular remodeling independent of its effect on blood pressure.

We examined whether overproduction of endogenous nitric oxide (NO) can prevent hypoxia-induced pulmonary hypertension and vascular remodeling by using endothelial NO-overexpressing (eNOS-Tg) mice. Male eNOS-Tg mice and their littermates (wild-type, WT) were maintained in normoxic or 10% hypoxic condition for 3 weeks. In normoxia, eNOS protein levels, Ca 2+ -dependent NOS activity, and cGMP levels in the lung of eNOS-Tg mice were higher than those of WT mice. Activity of eNOS and cGMP production in the lung did not change significantly by hypoxic exposure in either genotype. Chronic hypoxia did not induce iNOS expression nor increase its activity in either genotype. Plasma and lung endothelin-1 levels were increased by chronic hypoxia, but these levels were not significantly different between the 2 genotypes. In hemodynamic analysis, right ventricular systolic pressure (RVSP) in eNOS-Tg mice was similar to that in WT mice in normoxia. Chronic hypoxia increased RVSP and induced right ventricular hypertrophy in both genotypes; however, the degrees of these increases were significantly smaller in eNOS-Tg mice. Histological examination revealed that hypoxic mice showed medial wall thickening in pulmonary arteries. However, the increase of the wall thickening in small arteries (diameter

Abstract —NO produced by endothelial NO synthase (eNOS) plays important roles in the regulation of vascular tone and structure. The purpose of this study was to clarify the role of eNOS-derived NO on vascular remodeling by use of eNOS-transgenic (eNOS-Tg) mice. The common carotid artery was ligated just proximal to the carotid bifurcation. Four weeks later, the proximal carotid artery of the ligation site was histologically examined. In this vascular remodeling model, the endothelium remains uninjured, but neointimal and medial thickening occurs in combination with a reduction in vascular diameter at the proximal portion of the ligation. At 4 weeks after ligation, the respective neointimal and medial areas in wild-type mice were 17 200±1100 and 24 300±1500 μm 2 , whereas both were reduced to 8000±1900 ( P 2 ( P N G -Nitro- l -arginine methyl ester treatment increased neointimal and medial areas to the same extent in both genotypes. Leukocyte infiltration was observed in the luminal side of the vessel, but the number of infiltrating cells was significantly attenuated in eNOS-Tg mice compared with wild-type mice. This reduction of leukocyte infiltration in eNOS-Tg mice was associated with reduced expressions of intracellular adhesion molecule-1 and vascular cellular adhesion molecule-1 on the endothelium. In conclusion, chronic eNOS overexpression in the endothelium reduced leukocyte infiltration and inhibited neointimal formation and medial thickening. Our data provide the evidence for the regulatory role of NO from the endothelium on vascular structure integrity.

Studies in vitro reveal that fluvastatin, an HMG-CoA reductase inhibitor, has a strong DPPH radical scavenging activity and achieves concentration-dependent inhibition of copper- and cell-induced oxidation of low-density lipoprotein (LDL). To further examine the anti-oxidative activity of fluvastatin in vivo, we elucidated the effects of chronic treatment with fluvastatin at a dose insufficient to reduce plasma cholesterol levels (2 mg/kg per day) on vasomotion and vascular oxidative stress in thoracic aortas of 0.5% cholesterol-fed rabbits. After 12 weeks of dietary treatment, aortic segments from rabbits fed cholesterol alone showed impaired endothelium-dependent relaxation responses to acetylcholine and A23187 compared to normal chow-fed rabbits in association with a significant increase in plasma total cholesterol levels. In contrast, although plasma total cholesterol levels were not different from those in control cholesterol-fed rabbits, aortic segments from fluvastatin-treated rabbits showed normal relaxation. Compared with rabbits fed cholesterol alone, fluvastatin treatment decreased susceptibility of LDL to ex vivo copper-induced oxidation, reduced vascular superoxide generation, and atheromatous plaque formation. In conclusion, the potent anti-oxidative properties of fluvastatin in addition to its cholesterol-lowering activity appear to contribute to its anti-atherosclerotic effect in vivo.

Abstract —Lysophosphatidylcholine (lysoPC), a major lipid component of oxidized low density lipoprotein, inhibits endothelial cell (EC) migration and proliferation, which are critical processes during angiogenesis and the repair of injured vessels. However, the mechanism(s) of lysoPC-induced inhibition of EC migration and proliferation has not been clarified. In this report, we demonstrate the critical role of extracellular signal–regulated kinase (ERK) in growth factor–stimulated EC migration and proliferation as well as their inhibition by lysoPC. EC migration and proliferation stimulated by basic fibroblast growth factor (FGF-2) were blocked by inhibition of ERK activity by both the specific mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 and the overexpression of a dominant-negative mutant of MEK1. Conversely, overexpression of a constitutively active mutant of MEK1 increased EC migration and proliferation, which were comparable to those of ECs stimulated with FGF-2. LysoPC inhibited FGF-2–induced ERK activation via prevention of Ras activation without inhibiting tyrosine phosphorylation of phospholipase C-γ. Taken together, our data demonstrate that ERK activity is required for FGF-2–induced EC migration and proliferation and suggest that inhibition of the Ras/ERK pathway by lysoPC contributes to the reduced EC migration and proliferation.

Nitric oxide (NO), constitutively produced by endothelial nitric oxide synthase (eNOS), plays a major role in the regulation of blood pressure and vascular tone. We generated transgenic mice overexpressing bovine eNOS in the vascular wall using murine preproendothelin-1 promoter. In transgenic lineages with three to eight transgene copies, bovine eNOS-specific mRNA, protein expression in the particulate fractions, and calcium-dependent NOS activity were confirmed by RNase protection assay, immunoblotting, and L-arginine/citrulline conversion. Immunohistochemical studies revealed that eNOS protein was predominantly localized in the endothelial cells of aorta, heart, and lung. Blood pressure was significantly lower in eNOS-overexpressing mice than in control littermates. In the transgenic aorta, basal NO release (estimated by Nomega-nitro-L-arginine-induced facilitation of the contraction by prostaglandin F2alpha) and basal cGMP levels (measured by enzyme immunoassay) were significantly increased. In contrast, relaxations of transgenic aorta in response to acetylcholine and sodium nitroprusside were significantly attenuated, and the reduced vascular reactivity was associated with reduced response of cGMP elevation to these agents as compared with control aortas. Thus, our novel mouse model of chronic eNOS overexpression demonstrates that, in addition to the essential role of eNOS in blood pressure regulation, tonic NO release by eNOS in the endothelium induces the reduced vascular reactivity to NO-mediated vasodilators, providing several insights into the pathogenesis of nitrate tolerance.

Maternal immunization with oxidized lipoproteins prior to pregnancy protects against atherogenic in utero programming by gestational hypercholesterolemia and enhances beneficial lymphocyte-dependent immune responses in offspring. To determine whether in utero programming and immunomodulation also affect insulin resistance (IR) and type 2 diabetes, we investigated the effects of immunization on glucose and insulin responses in LDL receptor-deficient mice fed regular or 60% sucrose diets, as well as in offspring fed 0.5% cholesterol or 60% sucrose diets. IR was assessed by fasting glucose and insulin levels, oral glucose tolerance tests, glucose clamps, pancreatic immunohistochemistry and plasma free fatty acid concentrations. Immunizations improved glucose responses in both genders and protected both immunized mice and their offspring against IR and type 2 diabetes. Protection occurred even under euglycemic conditions, but was greatest in obese males exposed to very obesogenic/diabetogenic conditions. Hyperinsulinemic euglycemic clamps confirmed that maternal immunization protected mainly by reducing IR, but pancreatic immunocytochemistry also indicated some protection against beta cell damage. Maternal immunization was associated with marked regulation in offspring of 4 genes relevant to diabetes and 19 genes of importance for oxidative stress, as well as increased hepatic activities of key antioxidant enzymes. These findings establish that targeted immunomodulation may be used to protect immunized subjects and their offspring against IR and type 2 diabetes, and thus to reduce cardiovascular risk. They also support the notion that in utero programming influences offspring disease not by a single mechanism, but by multiple systemic effects.

Objective Maternal immunization with oxidized low-density lipoprotein prior to pregnancy prevents pathogenic in utero programming by gestational hypercholesterolemia, but it is unknown whether gestational hypercholesterolemia and maternal immunization affect similar pathways. Study Design A lipidomic approach was used for unbiased plasma eicosanoid profiling in adult offspring of immunized and nonimmunized normocholesterolemic or hypercholesterolemic rabbit mothers. Results Gestational hypercholesterolemia was associated with increased levels of some eicosanoids formed by the cyclooxygenase and 12-lipoxygenase pathways only (including thromboxane B 2 , prostaglandin [PG] F 2α , PGE 2 , and PGD 2 ). Immunization of hypercholesterolemic or normocholesterolemic mothers reduced 9 of 14 eicosanoids of the cyclooxygenase pathway, 21 of 23 eicosanoids of the 5- and 12-lipoxygenase pathways (eg, 5-hydroxyeicosatetraenoic acid, hepoxilin B 3 , 12-hydroxyeicosatetraenoic acid), 8 of 19 eicosanoids of the cytochrome P-450 pathway, and all metabolites of the nonenzymatic pathway. Conclusion Maternal immunization not only counteracts in utero programming by gestational hypercholesterolemia but reduces a broad range of eicosanoid modulators of immunity and inflammation in offspring.

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.

Prostaglandin E1 (PGE1) caused a rapid and dose-dependent increase in cAMP levels, followed by elevation of c-myc mRNA levels and then increased DNA synthesis in quiescent cultures of Swiss 3T3 fibroblasts. The dose-response curves of PGE1 were nearly the same for each of these three processes. Both 8-bromo-cAMP and forskolin increased c-myc mRNA levels to 40-50% and DNA synthesis to 70-80% of those caused by a maximally effective dose of PGE1. Under the comparable conditions, PGE1 did not stimulate diacylglycerol formation or activate protein kinase C. However, PGE1 did elevate cytoplasmic free Ca2+ concentration as measured with the fluorescent Ca2+ indicator quin 2. 8-Bromo-cAMP and forskolin were inactive in this capacity. The Ca2+ ionophore A23187 increased the level of c-myc mRNA. Diacylglycerol and Ca2+ mediate the elevation of c-myc mRNA levels which is caused by platelet-derived growth factor and fibroblast growth factor (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., and Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192). In contrast, the present results suggest that both cAMP and Ca2+ are involved in this PGE1-induced response in Swiss 3T3 cells.

At least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 were extracted from the crude membrane fraction of bovine brain by sodium cholate and purified by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite, and Mono Q HR5/5. One G protein with a Mr of about 24,000 (24K G) was purified to near homogeneity and characterized. 24K G bound maximally about 0.7 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 46 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. 24K G hydrolyzed GTP to liberate Pi with a rate of about 40 mmol of Pi/mol of protein/min. 24K G did not associate with the beta gamma subunit of Go. 24K G was distinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the ras protein (ras p21) and ADP-ribosylation factor, the G protein with a Mr of about 21,000 serving as a cofactor for the cholera toxin-dependent ADP-ribosylation of Gs. 24K G was not recognized by the antibody against ras p21, ADP-ribosylation factor, the alpha subunit each of Gs, Gi, and Go, or tubulin. These results indicate that 24K G is a novel G protein and suggest that there are multiple forms of G proteins with small Mr values as is the case with the G proteins which have the alpha subunits with Mr values of about 40,000.

A chromogenic substrate, H-D-Phe-Pip-Arg-pNA (S-2238) is a highly specific substrate to thrombin and releases p-nitroaniline (pNA) by the action of thrombin. We describe new modified APTT and PT methods using S-2238 in combination with the diazotization of pNA. In the modified APTT method, 100 microliter citrated plasma (diluted to 10-fold), 90 microliter 1 mM S-2238, 100 microliter 20 mM CaCl2 and 100 microliter Actin were mixed in an ice-bath, then incubated for 8 min at 37 degrees C. The reaction was stopped, and the generated pNA was diazotized by adding the following solutions sequentially: 975 microliter 0.04% sodium nitrite, 975 microliter 0.3% ammonium sulfamate and 975 microliter 0.07% N-(l-naphthyl)-ethylenediamine dihydrochloride. Diazotization changed pNA from yellow to pink. Then, absorbance at 545 nm was read, and values were expressed as thrombin units/ml plasma. In the modified PT method, 100 microliter citrated plasma (diluted to 20-fold), 90 microliter 1 mM S-2238 and 200 microliter tissue thromboplastin-C solution were mixed and processed as above. Correlations of the present modified APTT and APTT methods, and of modified PT and PT methods were significant (r = 0.426, p less than 0.01 and r = 0.561, p less than 0.01, respectively).

The addition of platelet-derived growth factor and fibroblast growth factor to quiescent cultures of Swiss 3T3 fibroblasts rapidly induced protein kinase C activation and Ca2+ mobilization and afterwards markedly increased c-myc mRNA levels. 1-Oleoyl-2-acetylglycerol, a membrane-permeable synthetic diacylglycerol, and 12-O-tetradecanoylphorbol 13-acetate, a tumor-promoting phorbol ester, stimulated protein kinase C activation without Ca2+ mobilization. Inversely, Ca2+ ionophores, A23187 and ionomycin, elicited Ca2+ mobilization without protein kinase C activation. Both protein kinase C-activating and Ca2+-mobilizing agents were able to increase c-myc mRNA levels in an additive manner. Prolonged treatment of the cells with phorbol 12,13-dibutyrate, another protein kinase C-activating phorbol ester, led to the down-regulation and complete disappearance of protein kinase C. In these cells, 1-oleoyl-2-acetylglycerol and 12-O-tetradecanoylphorbol 13-acetate did not increase c-myc mRNA levels, but platelet-derived growth factor, fibroblast growth factor, and the Ca2+ ionophores, all of which still induced Ca2+ mobilization, stimulated the increase of c-myc mRNA levels. These results strongly suggest that both protein kinase C and Ca2+ may be involved in platelet-derived growth factor- as well as fibroblast growth factor-induced expression of the c-myc oncogene in Swiss 3T3 cells.