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Bachground: Postprandial hyperlipaemia impairs endothelial function, possibly via oxidative-stress-mediated mechanisms. The aim of this study was to evaluate the acute effects of an oral triglyceride load (OTGL) on peripheral endothelial function in heart failure patients with reduced ejection fraction (HFrEF) compared to healthy controls., Design: Prospective cross-sectional., Methods: We enrolled 47 patients with HFrEF and 20 healthy controls. Peripheral endothelial function was assessed with EndoPAT2000 technology using a reactive hyperaemia index (RHI) and pulse wave amplitude (PWA) at baseline (after 8-h overnight fasting) as well as 1, 2, 3 and 4-h post-OTGL consumption (250-ml cream drink). Pulse wave amplitude index (PWAI) was calculated as a ratio of PWA at each time point to the baseline PWA., Results: RHI at baseline was lower in HFrEF patients compared to controls (1.7 ± 0.3 and 2.3 ± 0.6, respectively; P = 0.001). The OTGL accounted for a physiologic increase in PWA in healthy controls (p = 0.01), but this change was not observed in HFrEF patients. After 4 h, vasodilator response was significantly increased in healthy controls but not patients with HFrEF (2.3 ± 1.3 vs. 1.3 ± 0.8 respectively, P < 0.05)., Conclusions: The main finding of this study was the impaired postprandial dynamic changes in peripheral endothelial function in patients with HFrEF compared to healthy controls. A high-fat load that caused acute hypertriglyceridaemia significantly increased resting blood flow and peak flow at reactive hyperaemia in healthy subjects. By contrast, patients with HFrEF exhibited impaired dynamic changes in peripheral endothelial function after oral triglyceride load., Competing Interests: Declaration of Competing Interest On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2020 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

The mechanisms of the harmful cardiovascular effects of small particulate matter are incompletely understood. Endothelial progenitor cells (EPCs) predict outcome of patients with vascular disease. The aim of our study was to examine the effects of diesel exhaust particles (DEP) on EPC and on the associated vascular damage in mice. C57Bl/6 mice were exposed to DEP. 2 μg DEP/day was applicated intranasally for 3 weeks. Exposure to DEP reduced DiLDL/lectin positive EPC to 58.4 ± 5.6% (p < 0.005). Migratory capacity was reduced to 65.8 ± 3.9% (p < 0.0001). In ApoE(-/-) mice, DEP application reduced the number of EPC to 75.6 ± 6.4% (p < 0.005) and EPC migration to 58.5 ± 6.8% (p < 0.005). Neoangiogenesis was reduced to 39.5 ± 14.6% (p < 0.005). Atherogenesis was profoundly increased by DEP treatment (157.7 ± 18.1% vs. controls, p < 0.05). In cultured human EPC, DEP (0.1-100 μg/mL) reduced migratory capacity to 25 ± 2.6% (p < 0.001). The number of colony-forming units was reduced to 8.8 ± 0.9% (p < 0.001) and production of reactive oxygen species was elevated by DEP treatment (p < 0.001). Furthermore, DEP treatment increased apoptosis of EPC (to 266 ± 62% of control, p < 0.05). In a blood-brain barrier model, DEP treatment impaired endothelial cell integrity during oxygen-glucose deprivation (p < 0.001). Diesel exhaust particles impair endothelial progenitor cell number and function in vivo and in vitro. The reduction in EPC was associated with impaired neoangiogenesis and a marked increase in atherosclerotic lesion formation.

Background and Purpose: Chronic stress is associated with increased stroke risk. However, the underlying pathophysiological mechanisms are poorly understood. We examined the effects of chronic stress on endothelial function and ischemic brain injury in a mouse model., Methods: 129/SV mice were treated with glucocorticoid receptor antagonist mifepristone (25 mg kg(-1)/d) or vehicle and exposed to 28 days of chronic stress consisting of exposure to rat, restraint stress, and tail suspension. Heart rate and blood pressure were continuously recorded by telemetry. Endothelial nitric oxide synthase mRNA and protein expression as well as superoxide production and lipid hydroperoxides were quantified. Endothelium-dependent vasorelaxation was measured in aortic rings. Ischemic lesion volume was quantified after 30 minutes filamentous middle cerebral artery occlusion and 72 hours reperfusion., Results: Chronic stress caused a significant increase in heart rate, impaired endothelium-dependent vasorelaxation, increased superoxide production, and reduced aortic and brain endothelial nitric oxide synthase levels. Animals exposed to chronic stress showed major increases in ischemic lesion size. These deleterious effects of stress were completely reversed by treatment with mifepristone., Conclusions: Chronic stress increases stroke vulnerability likely through endothelial dysfunction, which can be reversed by a glucocorticoid receptor antagonist.

Background and Purpose: Vascular effects of mental stress are only partially understood. Therefore, we studied effects of chronic stress and heart rate (HR) on endothelial function and cerebral ischemia., Methods: 129S6/SvEv mice were randomized to the I(f)-channel inhibitor ivabradine (10 mg/kg per day) or vehicle and underwent a chronic stress protocol for 28 days., Results: Stress increased HR from 514 ± 10 bpm to 570 ± 14 bpm, this was prevented by ivabradine (485 ± 7 bpm). Endothelium-dependent relaxation of aortic rings was impaired in mice exposed to stress. HR reduction restored endothelial function to the level of naive controls. Vascular lipid hydroperoxides were increased to 333% ± 24% and vascular NADPH oxidase activity was upregulated to 223 ± 38% in stressed mice, which was prevented by ivabradine. Stress reduced aortic endothelial nitric oxide synthase mRNA expression to 84% ± 3% and increased AT1 receptor mRNA to 168% ± 18%. Both effects were attenuated by HR reduction. In brain tissue, stress resulted in an upregulation of lipid hydroperoxides to 140% ± 11%, which was attenuated by HR reduction. Ivabradine increased brain capillary density in naive and in stressed mice. Mice exposed to chronic stress before induction of ischemic stroke by transient middle cerebral artery occlusion exhibited increased lesion size (33.7 ± 2.3 mm3 versus 23.9 ± 2.4 mm3). HR reduction led to a marked reduction of the infarct volume to 12.9 ± 3.3 mm3., Conclusions: Chronic stress impairs endothelial function and aggravates ischemic brain injury. HR reduction protects from cerebral ischemia via improvement of endothelial function and reduction of oxidative stress. These results identify heart rate as a mediator of vascular effects induced by chronic stress.

Objective: To determine the effect of heart rate reduction with ivabradine on atherosclerotic lesions and erectile dysfunction., Methods: Two different treatment regimes with ivabradine were applied in wild-type (C57/B6) and ApoE(-/-)-mice to study effects of ivabradine on erectile function and atherosclerosis in animals with and without present endothelial dysfunction. Preventive effects of ivabradine were evaluated in animals fed a high-cholesterol diet in parallel to treatment with ivabradine (orally via chow, 10 mg/kg per day). The other treatment regime started treatment with a high-cholesterol diet for 4 weeks to induce endothelial dysfunction. Thereafter, treatment with ivabradine (orally via chow, 15 mg/kg per day) was started in ApoE(-/-) mice for 3 months. Vital parameters were measured using the tail-cuff method. Erectile function was assessed by pharmacological stimulation of corpora cavernosa in organ bath chambers. Atherosclerotic plaque formation, oxidative stress, eNOS and collagen content were determined., Results: Treatment with ivabradine significantly reduced heart rate (p<0.01), with no effect on blood pressure. Aortic atherosclerotic lesion size decreased with ivabradine in both treatment regimes (p<0.05). Endothelium-dependent relaxation of corpora cavernosa significantly decreased in ApoE(-/-)-mice with a restoration by ivabradine in prevention and reversal. Dihydroethidium-stained penile sections (p<0.05) and lipid peroxidase assay (p<0.05) revealed a reduction in superoxide production in ivabradine-treated animals. Penile eNOS-expression increased and collagen content significantly decreased (p<0.01) in ivabradine-treated animals., Conclusion: Ivabradine improves penile endothelial function by reduction of oxidative stress and penile fibrosis. Beneficial effects were achieved in prevention and manifest endothelial dysfunction., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Aims: Inhibition of the angiotensin-converting enzyme (ACE) prevents maladaptive cardiac remodelling. Endothelial progenitor cells (EPC) from the bone marrow contribute to endothelial repair and neovascularization, effects that are potentially important during cardiac remodelling. We hypothesized that ACE inhibitors may exert beneficial effects during pressure-induced myocardial hypertrophy by regulating progenitor cell function., Methods and Results: In C57/Bl6 mice, development of cardiac hypertrophy induced by transaortic constriction (TAC) for 5 weeks was reduced by ramipril, 5 mg/kg p.o., independent of blood pressure lowering. Ramipril prevented TAC-induced apoptosis of cardiac myocytes and endothelial cells. On day 1 after TAC, upregulation of Sca-1(pos)/KDR(pos) EPC was observed, which was further increased by ramipril. EPC were persistently elevated in the TAC mice receiving vehicle treatment but not in the ramipril group after 5 weeks. These effects were independent of hypoxia-inducible factor-1alpha mRNA and protein expression. The ACE inhibitor but not TAC improved the migratory capacity of DiLDL(pos) EPC. Increased cardiac afterload induced upregulation of extracardiac neoangiogenesis. This effect was enhanced by ACE inhibition. Ramipril but not TAC markedly increased cardiac capillary density determined by the ratio of CD31(pos) cells to cardiomyocytes. Bone marrow transplantation studies revealed that TAC increased the percentage of bone marrow-derived GFP(pos) endothelial cells in the myocardium, and ramipril made this effect more pronounced., Conclusions: ACE inhibition prevents pressure-induced maladaptive cardiac hypertrophy and increases intra- and extracardiac neoangiogenesis associated with the upregulation of EPC and amelioration of EPC migration. The regulation of progenitor cells from the bone marrow identifies a novel effect of ACE inhibitors during cardiac remodelling.

HMG CoA reductase inhibitors (statins) have been shown to be effective lipid lowering agents and are beneficial in the primary and secondary prevention of coronary heart disease. However, the overall benefits observed with statins appear to be greater than what might be expected from changes in lipid levels alone and the positive effects have only partially been reproduced with other lipid lowering drugs, suggesting effects in addition to cholesterol lowering. In experimental models, many of the cholesterol-independent effects of statins are mediated by inhibition of isoprenoids, which serve as lipid attachments for intracellular signalling molecules such as small Rho guanosine triphosphate-binding proteins, whose membrane localization and function are dependent on isoprenylation. This review summarizes the effects of statins on endothelial function and oxidative stress.

Background: Elevated heart rate is associated with increased cardiovascular morbidity. We hypothesized that selective heart rate reduction may influence endothelial function and atherogenesis and tested the effects of the I(f) current inhibitor ivabradine in apolipoprotein E-deficient mice., Methods and Results: Male apolipoprotein E-deficient mice fed a high-cholesterol diet were treated with ivabradine (10 mg . kg(-1) . d(-1)) or vehicle for 6 weeks (n=10 per group). Ivabradine reduced heart rate by 13.4% (472+/-9 versus 545+/-11 bpm; P<0.01) but did not alter blood pressure or lipid levels. Endothelium-dependent relaxation of aortic rings was significantly improved in ivabradine-fed animals (P<0.01). Ivabradine decreased atherosclerotic plaque size in the aortic root by >40% and in the ascending aorta by >70% (P<0.05). Heart rate reduction by ivabradine had no effect on the number of endothelial progenitor cells and did not alter aortic endothelial nitric oxide synthase, phosphorylated Akt, vascular cell adhesion molecule-1, or intercellular adhesion molecule-1 expression but decreased monocyte chemotactic protein-1 mRNA and exerted potent antioxidative effects. Ivabradine reduced vascular NADPH oxidase activity to 48+/-6% and decreased markers of superoxide production and lipid peroxidation in the aortic wall (P<0.05). The in vivo effects of ivabradine were absent at a dose that did not lower heart rate, in aortic rings treated ex vivo, and in cultured vascular cells. In contrast to ivabradine, treatment with hydralazine (25 mg . kg(-1) . d(-1) for 6 weeks) reduced blood pressure (-15%) but increased heart rate (37%) and did not improve endothelial function, atherosclerosis, or oxidative stress., Conclusions: Selective heart rate reduction with ivabradine decreases markers of vascular oxidative stress, improves endothelial function, and reduces atherosclerotic plaque formation in apolipoprotein E-deficient mice.

Objective: Peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists (thiazolidinediones [TZDs]) are used for the treatment of diabetes. Bone marrow-derived endothelial progenitor cells (EPCs) improve vascular function and predict cardiovascular risk. The effect of pioglitazone therapy on EPCs was examined., Research Design and Methods and Results: We performed a prospective, randomized, double-blind study on patients with documented stable coronary artery disease and normal glucose tolerance. Of 54 patients with normal fasting glucose levels, 18 showed impaired glucose tolerance and 36 patients with normal glucose tolerance were randomized to 30-day treatment with pioglitazone (45 mg) or placebo in addition to optimal medical therapy. All patients in the TZD group showed an increase of adiponectin levels as an indicator of compliance (11.4 +/- 1.1 to 36.8 +/- 2.1 microg/ml; P < 0.001). TZD, but not placebo, decreased mean high-sensitivity C-reactive protein to 43 +/- 19% (P < 0.05). Pioglitazone increased CD34(+)/kinase insert domain receptor(+) EPCs to 142 +/- 9% and cultured 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated LDL(+)/lectin(+) EPCs to 180 +/- 3% (P < 0.05). EPC numbers were not changed in the placebo group. TZD increased the SDF-1-induced migratory capacity to 146 +/- 9% per EPC number (P < 0.05) and upregulated the clonogenic potential of EPCs, increasing the colony-forming units to 172 +/- 12% (P < 0.001). In cultured human EPCs, TZD increased EPC numbers and migration and reduced NADPH-oxidase activity. The TZD effect was reversed by the PPAR gamma antagonist GW9662 and mimicked by treatment with adiponectin., Conclusions: The PPAR gamma agonist pioglitazone increases the number and function of EPCs in patients with coronary artery disease. The effect represents a potential regenerative mechanism in atherosclerosis and is observed in normoglycemic individuals with stable coronary artery disease.

Objective: Sedentary lifestyle is associated with increased cardiovascular events. The underlying molecular mechanisms are incompletely understood. Reactive oxygen species (ROS) contribute to endothelial dysfunction and atherosclerosis. An important source of vascular ROS is the NADPH oxidase., Methods and Results: C57BL6 mice were subjected to regular housing (physical inactivity) or voluntary training on running wheels (6 weeks). Inactivity increased vascular lipid peroxidation to 148+/-9% and upregulated superoxide release to 176+/-17% (L-012 chemiluminescence) and 188+/-29% (cytochrome C reduction assay), respectively. ROS production was predominantly increased in the endothelium and the media (dihydroethidium fluorescence). Activity of the NADPH oxidase was increased to 154+/-22% in the sedentary group. Rac1 GST-PAK pull-down assays showed an upregulation of rac1 activity to 161+/-14%. Expression levels of the subunits nox1, p47phox, and p67phox were increased. To address the significance of the antioxidative effects of running, experiments were repeated in apolipoprotein E-deficient mice treated with a high-cholesterol diet. Inactivity increased vascular superoxide production and impaired endothelium-dependent vasorelaxation. Atherosclerotic lesion formation was significantly accelerated in sedentary mice., Conclusions: Inactivity increases vascular NADPH oxidase expression and activity and enhances vascular ROS production, which contributes to endothelial dysfunction and atherosclerosis during sedentary as opposed to physically active lifestyle.

Statins are potent inhibitors of cholesterol biosynthesis. In clinical trials, statins are beneficial in the primary and secondary prevention of coronary heart disease. However, the overall benefits observed with statins appear to be greater than what might be expected from changes in lipid levels alone, suggesting effects beyond cholesterol lowering. Indeed, recent studies indicate that some of the cholesterol-independent or "pleiotropic" effects of statins involve improving endothelial function, enhancing the stability of atherosclerotic plaques, decreasing oxidative stress and inflammation, and inhibiting the thrombogenic response. Furthermore, statins have beneficial extrahepatic effects on the immune system, CNS, and bone. Many of these pleiotropic effects are mediated by inhibition of isoprenoids, which serve as lipid attachments for intracellular signaling molecules. In particular, inhibition of small GTP-binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation, may play an important role in mediating the pleiotropic effects of statins.

Endothelium dysfunction may result from increased production of reactive oxygen species and decreased availability of nitric oxide. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (ie, statins) exert cholesterol-independent vasoprotective effects that are mediated, in part, through the inhibition of small G-proteins Rho and Rac. Rho negatively regulates endothelial nitric oxide synthase and Rac contributes to NAD(P)H-oxidase activation and superoxide production. Statins inhibit both Rho and Rac GTPase activity via inhibition of geranylgeranylation, which confers endothelial nitric oxide synthase upregulation and decreases superoxide production, respectively. Sudden discontinuation of statin therapy may have negative effects. Withdrawal of statin treatment leads to an overshoot activation of Rho and Rac with dramatic effects on nitric oxide bioavailability, NAD(P)H-oxidase activity, and superoxide production.

Background: The molecular mechanisms by which physical training improves peripheral and coronary artery disease are poorly understood. Bone marrow-derived endothelial progenitor cells (EPCs) are thought to exert beneficial effects on atherosclerosis, angiogenesis, and vascular repair., Methods and Results: To study the effect of physical activity on the bone marrow, EPCs were quantified by fluorescence-activated cell sorter analysis in mice randomized to running wheels (5.1+/-0.8 km/d, n=12 to 16 per group) or no running wheel. Numbers of EPCs circulating in the peripheral blood of trained mice were enhanced to 267+/-19%, 289+/-22%, and 280+/-25% of control levels after 7, 14, and 28 days, respectively, accompanied by a similar increase of EPCs in the bone marrow and EPCs expanded from spleen-derived mononuclear cells. eNOS-/- mice and wild-type mice treated with N(G)-nitro-l-arginine methyl ester showed lower EPC numbers at baseline and a significantly attenuated increase of EPC in response to physical activity. Exercise NO dependently increased serum levels of vascular endothelial growth factor and reduced the rate of apoptosis in spleen-derived EPCs. Running inhibited neointima formation after carotid artery injury by 22+/-2%. Neoangiogenesis, as assessed in a subcutaneous disc model, was increased by 41+/-16% compared with controls. In patients with stable coronary artery disease (n=19), moderate exercise training for 28 days led to a significant increase in circulating EPCs and reduced EPC apoptosis., Conclusions: Physical activity increases the production and circulating numbers of EPCs via a partially NO-dependent, antiapoptotic effect that could potentially underlie exercise-related beneficial effects on cardiovascular diseases.

Endothelial cell damage is one important pathophysiological step of atherosclerosis and restenosis after angioplasty. Accelerated reendothelialization impairs neointima formation. We evaluated the role of intravenously transfused endothelial progenitor cells (EPCs) on reendothelialization and neointima formation in a mouse model of arterial injury. Spleen-derived mouse mononuclear cells (MNCs) were cultured in endothelial basal medium. A total of 91.8+/-3.2% of adherent cells showed uptake of acetylated low-density lipoprotein (Dil-Ac-LDL) and lectin binding after 4 days. Immunostaining and long-term cultures confirmed the endothelial progenitor phenotype. To determine the effect of stem cell transfusion on reendothelialization, mice received either fluorescent-labeled spleen-derived MNCs or in vitro differentiated EPCs intravenously after endothelial injury of the carotid artery. Transfused cells were strictly restricted to the injury site, and lectin binding confirmed the endothelial phenotype. Homing of transfused cells to the site of injury was only detectable in splenectomized mice. Cell transfusion caused enhanced reendothelialization associated with a reduction of neointima formation. Systemically applied spleen-derived MNCs and EPCs home to the site of vascular injury, resulting in an enhanced reendothelialization associated with decreased neointima formation. These results allow novel insights in stem cell biology and provide additional information for the treatment of vascular dysfunction and prevention of restenosis after angioplasty. The full text of this article is available online at http://www.circresaha.org.

Endothelial dysfunction is now recognised as an important process in the pathogenesis of atherosclerosis. Nitric oxide (NO) release by the endothelium regulates blood flow, inflammation and platelet aggregation, and consequently its disruption during endothelial dysfunction can decrease plaque stability and encourage the formation of atherosclerotic lesions and thrombi. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) are often utilised in the prevention of coronary heart disease due to their efficacy at lowering lipid levels. However, statins may also prevent atherosclerotic disease by non-lipid or pleiotropic effects, for example, improving endothelial function by promoting the production of NO. There are various mechanisms whereby statins may alter NO release, such as inhibiting the production of mevalonate and important isoprenoid intermediates, thereby preventing the isoprenylation of the small GTPase Rho, which negatively regulates the expression of endothelial nitric oxide synthase (eNOS). Furthermore, statins may also increase eNOS activity via post-translational activation of the phosphatidylinositol 3-kinase/protein kinase Akt (PI3 K/Akt) pathway and/or through an interaction with the molecular chaperone heat-shock protein 90 (HSP90). Data suggest that statins may vary in their efficacy for enhancing the release of NO, and the mechanisms dictating these differences are not yet clear. By increasing NO production, statins may interfere with atherosclerotic lesion development, stabilise plaque, inhibit platelet aggregation, improve blood flow and protect against ischaemia. Therefore, the ability of statins to improve endothelial function through the release of NO may partially account for their beneficial effects at reducing the incidence of cardiovascular events.

Objective: Atherosclerosis and restenosis after vascular injury are both characterized by endothelial dysfunction, apoptosis, inappropriate endothelialization, and neointimal formation. Bone marrow-derived endothelial progenitor cells have been implicated in neovascularization, resulting in adult blood vessel formation. Despite the anticipated stem cell plasticity, the role of bone marrow-derived endothelial progenitor cells has not been clarified in vascular lesion development., Methods and Results: We investigated vascular lesion formation in mice after transplantation of bone marrow transfected by means of retrovirus with enhanced green fluorescent protein. Carotid artery injury was induced, resulting in neointimal formation. Fluorescence microscopy and immunohistological analysis revealed that bone marrow-derived progenitor cells are involved in reendothelialization of the vascular lesions. Treatment with rosuvastatin (20 mg/kg body wt per day), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, enhanced the circulating pool of endothelial progenitor cells, propagated the advent of bone marrow-derived endothelial cells in the injured vessel wall, and, thereby, accelerated reendothelialization and significantly decreased neointimal formation., Conclusions: Vascular lesion development initiated by endothelial cell damage is moderated by bone marrow-derived progenitor cells. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibition promotes bone marrow-dependent reendothelialization and diminishes vascular lesion development. These findings may help to establish novel pathophysiological concepts and therapeutic strategies in the treatment of various cardiovascular diseases.

Objective: Hypercholesterolemia-induced angiotensin II type 1 (AT1) receptor overexpression is thought to be a key event in the development of endothelial dysfunction., Methods and Results: The effect of a 6-week treatment with the AT1 receptor antagonist candesartan (16 mg/d) on endothelial function and serum inflammation markers was compared with the effect of treatment with placebo or the calcium channel antagonist felodipine (5 mg/d) in 47 hypercholesterolemic patients (low density lipoprotein cholesterol >160 mg/dL). Endothelial function was assessed by measurement of forearm blood flow (FBF) by venous occlusion plethysmography. FBF during reactive hyperemia was significantly improved by candesartan, whereas felodipine and placebo exerted no effect. Nitroglycerin-induced vasorelaxation and basal FBF were not altered significantly. Blood pressure and cholesterol levels were not affected significantly by any drug. Serum concentrations of 8-isoprostane, monocyte chemoattractant protein-1, and soluble intercellular adhesion molecule-1 were significantly reduced by candesartan treatment but not by placebo or felodipine (ELISA assays). Levels of high-sensitivity C-reactive protein and tumor necrosis factor-alpha were not altered significantly by any treatment., Conclusions: These data suggest that AT1 receptor antagonism improves endothelial function during hypercholesterolemia and that this applies not only to endothelium-dependent vasodilatation but also to oxidative stress and events involved in monocyte attraction and adhesion. AT1 receptor blockade may potentially represent a novel approach for the prevention of vascular dysfunction associated with hypercholesterolemia that is independent of lipid-lowering and blood pressure-lowering interventions.

HMG-CoA reductase inhibitors (statins) are cholesterol-lowering drugs and reduce the risk of myocardial infarction and stroke. In this study we investigated whether rosuvastatin, a new, potent HMG-CoA reductase inhibitor, upregulates endothelial nitric oxide (NO) expression and activity and protects from cerebral ischaemia in mice. Endothelial cells in culture and 129/SV mice were chronically treated with rosuvastatin. The expression and activity of endothelial NO synthase (eNOS) was determined by reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting and arginine-citrulline assays. Cerebral ischaemia was induced by occlusion of the middle cerebral artery (MCAo) for 2 h and infarct size was determined after 22 h of reperfusion. Treatment of endothelial cells with rosuvastatin concentration- and time-dependently upregulated eNOS mRNA and protein expression. In aortas of 129/SV wild-type mice, treatment with 0.2, 2, and 20 mg kg(-1) rosuvastatin subcutaneously (s.c.) for 10 days significantly upregulated eNOS mRNA by 50, 142, and 205%, respectively. NOS activity was significantly increased by 75, 145, and 320%, respectively. Stroke volume after 2-h MCAo was reduced by 27, 56, and 50% (for 0.2, 2 and 20 mg kg(-1), respectively). Serum cholesterol and triglygeride levels were not significantly lowered by the treatment. The novel HMG-CoA reductase inhibitor rosuvastatin dose-dependently upregulates eNOS expression and activity and protects from cerebral ischaemia in mice. The effects are independent of changes in cholesterol levels and are equivalent or even superior to the protective effects by simvastatin and atorvastatin in this animal model.

Background: It has not been completely clarified whether selective estrogen receptor modulators (SERMs) such as raloxifene exert vasoprotective effects similar to those of estrogens., Methods and Results: To investigate vascular effects of raloxifene, male spontaneously hypertensive rats were treated for 10 weeks with either raloxifene (10 mg x kg(-1) x d(-1)) or vehicle. Raloxifene improved endothelium-dependent vasodilatation but had no effect on either endothelium-independent vasorelaxation or phenylephrine-induced vasoconstriction. Raloxifene treatment increased the release of NO from the vessel wall by enhanced expression and activity of endothelial NO synthase. Blood pressure reduction after bradykinin infusion was more pronounced in animals treated with SERMs. The production of superoxide in intact aortic segments was decreased by raloxifene treatment. Administration of raloxifene had no effect on the expression of the essential NAD(P)H oxidase subunits p22phox and nox1 in the vasculature but reduced the activity and expression of vascular membrane-bound rac1, a GTPase required for the activation of the NAD(P)H oxidase. Finally, blood pressure levels were significantly decreased in spontaneously hypertensive rats treated with raloxifene. All SERM effects were also detected in healthy age-matched Wistar rats. In cultured rat aortic vascular smooth muscle cells, raloxifene inhibited angiotensin II-induced reactive oxygen species production dependent on estrogen receptor activation., Conclusions: Raloxifene treatment improves hypertension-induced endothelial dysfunction by increased bioavailability of NO. This is achieved by an increased activity of endothelial NO synthase and by an estrogen receptor-dependent reduction in release of reactive oxygen species from vascular cells. These vascular effects cause a profound blood pressure reduction and lead to decreased vascular damage in male spontaneously hypertensive rats.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) significantly reduce cardiovascular mortality associated with hypercholesterolemia. There is evidence that statins exert beneficial effects in part through direct effects on vascular cells independent of lowering plasma cholesterol. We characterized the effect of a 30-day treatment with atorvastatin in normocholesterolemic, spontaneously hypertensive rats (SHR). Systolic blood pressure was significantly decreased in atorvastatin-treated rats (184+/-5 versus 204+/-6 mm Hg for control). Statin therapy improved endothelial dysfunction, as assessed by carbachol-induced vasorelaxation in aortic segments, and profoundly reduced angiotensin II-induced vasoconstriction. Angiotensin type 1 (AT(1)) receptor, endothelial cell NO synthase (ecNOS), and p22phox mRNA expression were determined with quantitative reverse transcription-polymerase chain reaction. Atorvastatin treatment downregulated aortic AT(1) receptor mRNA expression to 44+/-12% of control and reduced mRNA expression of the essential NAD(P)H oxidase subunit p22phox to 63+/-7% of control. Aortic AT(1) receptor protein expression was consistently decreased. Vascular production of reactive oxygen species was reduced to 62+/-12% of control in statin-treated SHR, as measured with lucigenin chemiluminescence assays. Accordingly, treatment of SHR with the AT(1) receptor antagonist fonsartan improved endothelial dysfunction and reduced vascular free-radical release. Moreover, atorvastatin caused an upregulation of ecNOS mRNA expression (138+/-7% of control) and an enhanced ecNOS activity in the vessel wall (209+/-46% of control). Treatment of SHR with atorvastatin causes a significant reduction of systolic blood pressure and a profound improvement of endothelial dysfunction mediated by a reduction of free radical release in the vasculature. The underlying mechanism could in part be based on the statin-induced downregulation of AT(1) receptor expression and decreased expression of the NAD(P)H oxidase subunit p22phox, because AT(1) receptor activation plays a pivotal role for the induction of this redox system in the vessel wall.

To determine whether nuclear factor (NF)-kappaB is necessary to confer endothelial cell responsiveness to interferon (INF)-gamma in terms of vascular cell adhesion molecule (VCAM)-1 expression and leukocyte adhesion, human endothelial cells were treated with IFN-gamma in the presence of low concentrations (LCs) of interleukin (IL)-1alpha (

Background: Statins improve endothelial function by upregulating endothelial nitric oxide (NO) production that is mediated by inhibiting the isoprenylation of rho GTPase. Withdrawal of statin treatment could suppress endothelial NO production and may impair vascular function., Methods and Results: To test this hypothesis, mice were treated for 14 days with 10 mg/kg atorvastatin per day; this led to the upregulation of endothelial NO synthase expression and activity by 2.3- and 3-fold, respectively. Withdrawal of statins resulted in a dramatic, 90% decrease of NO production after 2 days. In mouse aortas and cultured endothelial cells, statins upregulated the expression of rho GTPase in the cytosol, but statins blocked isoprenoid-dependent rho membrane translocation and GTP-binding activity. Inhibiting the downstream targets of rho showed that rho expression is controlled by a negative feedback mechanism mediated by the actin cytoskeleton. Measuring rho mRNA half-life and nuclear run-on assays demonstrated that statins or disruption of actin stress fibers increased rho gene transcription but not rho mRNA stability. Therefore, treatment with statins leads to the accumulation of nonisoprenylated rho in the cytosol. Withdrawing statin treatment restored the availability of isoprenoids and resulted in a massive membrane translocation and activation of rho, causing downregulation of endothelial NO production., Conclusions: Withdrawal of statin therapy in normocholesterolemic mice results in a transient increase of rho activity, causing a suppression of endothelial NO production. The underlying molecular mechanism is a negative feedback regulation of rho gene transcription mediated by the actin cytoskeleton.

Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-deficient; eNOS(-/-)) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion. We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion. Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 microgram/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity. This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 +/- 3 hours to 24 +/- 4 hours. Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression. Inhibition of vascular Rho guanosine-5'-triphosphate binding activity by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion. No neuroprotection was observed with these agents in eNOS(-/-) mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.

The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, or statins, are potent inhibitors of cholesterol synthesis and large clinical trials have demonstrated that these agents reduce cholesterol and the incidence of cardiovascular diseases. Recent evidence, however, suggests that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels. Because statins also inhibit the synthesis of isoprenoid intermediates in the cholesterol biosynthetic pathway, they may have pleiotropic effects on vascular wall cells. In particular, the small GTP-binding protein, Rho, whose membrane localization and activity are affected by post-translational isoprenylation, may play an important role in mediating the direct vascular effects of statins.

We investigated whether inflammatory cytokines or oxidized low density lipoproteins (Ox-LDL) present in human atheroma modulate extracellular matrix degradation by inducing membrane type 1-matrix metalloproteinase (MT1-MMP) expression. Cultured human endothelial cells (EC) constitutively expressed MT1-MMP mRNA and protein with enzymatic activity. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha, or interleukin-1beta caused a time-dependent increase in the steady-state MT1-MMP mRNA levels within 4 h of exposure, peaking about 4-fold by 6 h, and remaining elevated for 12 h. Increased MT1-MMP mRNA correlated with a 2.5-fold increase in MT1-MMP protein in EC membranes. Ox-LDL also increased MT1-MMP mRNA levels that varied with the duration of exposure and degree of LDL oxidation. The increase in MT1-MMP mRNA occurred within 6 h of exposure to Ox-LDL and peaked over 3-fold by 6 h. Ox-LDL, but not native LDL, increased MT1-MMP protein by 2-fold in EC membranes. A combination of TNF-alpha and Ox-LDL was additive in increasing MT1-MMP expression. Nuclear run-on assays showed that TNF-alpha or Ox-LDL augmented steady-state mRNA levels by increased transcription of the MT1-MMP gene. These findings indicate that activation of EC by inflammatory cytokines and/or Ox-LDL increase MT1-MMP expression. Since MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokines or Ox-LDL may influence extracellular matrix remodeling.

The mechanism by which moderate alcohol ingestion lowers the risk of cardiovascular disease is unknown but may be due, in part, to the ability of alcohol to increase the level of tissue-type plasminogen activator (t-PA). Human endothelial cells were treated with low concentrations of ethanol (0.25-25 mM, 0-24 h), which are associated with moderate alcohol consumption. Although treatment with ethanol alone did not affect t-PA gene transcription or mRNA expression, it augmented isoproterenol (ISO)-stimulated t-PA gene transcription and mRNA levels by 3.4- and 2.8-fold, respectively, and decreased plasminogen activator inhibitor-1 mRNA levels by 65%. These effects of ethanol correlated with 2.5- and 6.9-fold increases in ISO-stimulated cyclic AMP levels and 4x-cyclic AMP response element heterologous promoter activity, respectively. To determine whether alcohol-induced changes in agonist-stimulated cyclic AMP levels were because of modulation of guanine nucleotide-binding proteins (G proteins), we assessed the effects of ethanol on Galphas and Galphai2. Although ethanol did not affect the expression of Galphas or Galphai2, it increased ISO-stimulated Galphas GTPase and GTP binding activity by 2.2- and 2.9-fold and decreased UK14304-stimulated Galphai2 GTPase and GTP binding activity by 38 and 80%. These results indicate that treatment with relatively low concentrations of ethanol enhances agonist-stimulated cyclic AMP-dependent t-PA gene transcription in vascular endothelial cells through differential modulation of G protein.

Endothelial-derived nitric oxide (NO) is an important mediator of vascular function. Clinical studies indicate that HMG-CoA reductase inhibitors (statins) improve endothelial function and reduce the incidence of stroke and myocardial infarction. Treatment of human endothelial cells with statins increased the expression of endothelial NO synthase (eNOS) protein and mRNA expression. Statins increased eNOS mRNA half-life but did not change eNOS gene transcription. Inhibition of mevalonate synthesis by statins not only blocks the formation of cholesterol but also of isoprenoids. The upregulation of eNOS expression by statins was independent of cholesterol but mediated via the inhibition of the isoprenoid geranylgeraniol, whereas farnesiol had no effect on eNOS. Immunoblot analyses, (35S)-GTP gamma S-binding assays and transfection studies revealed that statins upregulate eNOS expression by blocking the geranylgeranylation of the GTPase Rho which is necessary for its membrane-associated activity. Studies with mice showed, that statin treatment upregulates eNOS expression and function independent of serum cholesterol levels. Prophylactic treatment with statins augmented cerebral blood flow and reduced cerebral infarcts in normocholesterolemic mice. These effects of statins were completely absent in eNOS-deficient mice indicating that enhanced eNOS activity by statins is the predominant mechanism by which these agents protect against cerebral injury. Our results suggest that statins provide a novel prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia. Upregulation of eNOS by inhibiting Rho may provide a new pharmacologic target for the treatment of arteriosclerosis, pulmonary hypertension, and heart failure.

The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.

Background: Oxidized low-density lipoprotein (ox-LDL) causes endothelial dysfunction in part by decreasing the availability of endothelial nitric oxide (NO). Although HMG CoA reductase inhibitors restore endothelial function by reducing serum cholesterol levels, it is not known whether they can also directly upregulate endothelial NO synthase (ecNOS) activity., Methods and Results: Human saphenous vein endothelial cells were treated with ox-LDL (50 microg/mL thiobarbituric acid reactive substances 12 to 16 nmol/mg) in the presence of HMG CoA reductase inhibitors simvastatin and lovastatin. In a time-dependent manner, ox-LDL decreased ecNOS mRNA and protein levels (91+/-4% and 67+/-8% reduction after 72 hours, respectively). Both simvastatin (1 micromol/L) and lovastatin (10 micromol/L) upregulated ecNOS expression by 3.8-fold and 3.6-fold, respectively, and completely prevented its downregulation by ox-LDL. These effects of simvastatin on ecNOS expression correlated with changes in ecNOS activity. Although L-mevalonate alone did not affect ecNOS expression, cotreatment with L-mevalonate completely reversed ecNOS upregulation by simvastatin. Actinomycin D studies revealed that simvastatin stabilized ecNOS mRNA (tau1/2, 43 versus 35 hours). Nuclear run-on assays and transient transfection studies with a -1.6 kb ecNOS promoter construct showed that simvastatin did not affect ecNOS gene transcription., Conclusions: Inhibition of endothelial HMG CoA reductase upregulates ecNOS expression predominantly by posttranscriptional mechanisms. These findings suggest that HMG CoA reductase inhibitors may have beneficial effects in atherosclerosis beyond that attributed to the lowering of serum cholesterol by increasing ecNOS activity.

Hypoxia induces vasoconstriction, in part, by down-regulating endothelial cell nitric oxide synthase (ecNOS) expression. Previous studies indicate that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors improve endothelium-dependent relaxation by increasing ecNOS activity. To determine whether HMG CoA reductase inhibitors can prevent hypoxia-mediated down-regulation of ecNOS function and expression, human endothelial cells were exposed to hypoxia (3% O2) in the presence of HMG CoA reductase inhibitors simvastatin and lovastatin for various durations (0-48 h). Hypoxia decreased ecNOS protein and mRNA levels in a time-dependent manner, resulting in a 4- and 9-fold reduction after 48 h, respectively. In a concentration-dependent manner, simvastatin, and to a lesser extent, lovastatin, prevented the down-regulation of ecNOS expression by hypoxia. Simvastatin-induced changes in ecNOS expression correlated with changes in endothelial NO production and were reversed by treatment with L-mevalonate. Actinomycin D studies revealed that under hypoxic conditions, simvastatin increased ecNOS mRNA half-life from 13 to 38 h. Nuclear run-on studies showed that simvastatin had no effect on repression of ecNOS gene transcription by hypoxia. These results indicate that HMG CoA reductase inhibitors regulate ecNOS function and expression through changes in ecNOS mRNA stability and suggest that treatment with HMG CoA reductase inhibitors may have beneficial effects in patients with hypoxia-mediated pulmonary hypertension.

Background and Purpose: In the carotid bifurcation, atherosclerotic plaques usually develop in the outer wall of the internal carotid artery. The aim of this study was to analyze the expression of the cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in the carotid bifurcation. These molecules play a role in inflammatory cell recruitment and atherosclerosis., Methods: We examined the expression of ICAM-1, VCAM-1, and E-selectin in 22 human carotid bifurcation specimens by means of immunohistochemistry. Double immunostaining was performed with antibodies against CD-3, CD-31, CD-68, and alpha-smooth muscle actin combined with each of the cell adhesion molecule antibodies. In situ hybridization for ICAM-1 was performed in selected specimens., Results: A profound focal expression of ICAM-1 in the outer wall of the internal carotid artery could be demonstrated (86% of all specimens). This focal expression could be shown in histologically normal appearing bifurcations of young adults. ICAM-1 was expressed by subsets of macrophages and smooth muscle cells and by endothelial cells. VCAM-1 and E-selectin showed no focal expression and were not found in normal carotids. In advanced plaques all three adhesion molecules-ICAM-1, VCAM-1, and E-selectin-were expressed., Conclusions: We were able to demonstrate a focal expression of ICAM-1 in the outer lateral wall of the internal carotid artery, which is a high-risk region for the development of atherosclerotic lesions.