Sven Wassmann

Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis.

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 NG-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.

HMG-CoA Reductase Inhibitors Improve Endothelial Dysfunction in Normocholesterolemic Hypertension via Reduced Production of Reactive Oxygen Species

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 (AT1) receptor, endothelial cell NO synthase (ecNOS), and p22phox mRNA expression were determined with quantitative reverse transcription-polymerase chain reaction. Atorvastatin treatment downregulated aortic AT1 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 AT1 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 AT1 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 AT1 receptor expression and decreased expression of the NAD(P)H oxidase subunit p22phox, because AT1 receptor activation plays a pivotal role for the induction of this redox system in the vessel wall.

Modulation of Antioxidant Enzyme Expression and Function by Estrogen

&NA; Oxidative stress plays a pivotal role in the pathogenesis of atherosclerosis and can be effectively influenced by radical scavenging enzyme activity and expression. The vasoprotective effects of estrogens may be related to antioxidative properties. Therefore, effects of 17&bgr;‐estradiol on production of reactive oxygen species and radical scavenging enzymes were investigated. 17&bgr;‐estradiol diminished angiotensin II‐induced free radical production in vascular smooth muscle cells (DCF fluorescence laser microscopy). 17&bgr;‐estradiol time‐ and concentration‐dependently upregulated manganese (MnSOD) and extracellular superoxide dismutase (ecSOD) expression (Northern and Western blotting) and enzyme activity (photometric assay). Nuclear run‐on assays demonstrated that 17&bgr;‐estradiol increases MnSOD and ecSOD transcription rate. Half‐life of MnSOD mRNA was not influenced, whereas ecSOD mRNA was stabilized by estrogen. Copper‐zinc SOD, glutathione‐peroxidase, and catalase were not affected by estrogen. Estrogen deficiency in ovariectomized mice induced a downregulation of ecSOD and MnSOD expression, which was associated with increased production of vascular free radicals and prevented by estrogen replacement or treatment with PEG‐SOD. In humans, increased estrogen levels led to enhanced ecSOD and MnSOD expression in circulating monocytes. Estrogen acts antioxidative at least to some extent via stimulation of MnSOD and ecSOD expression and activity, which may contribute to its vasoprotective effects. (Circ Res. 2003;93:170‐177.)

Circulating CD31+/Annexin V+ Apoptotic Microparticles Correlate With Coronary Endothelial Function in Patients With Coronary Artery Disease

Objective—Endothelial dysfunction predicts morbidity and mortality in patients at cardiovascular risk. Endothelial function may be decisively influenced by the degree of endothelial cell apoptosis. Methods and Results—To test this hypothesis in humans, endothelial-dependent vasodilatation was invasively assessed in 50 patients with coronary artery disease (CAD) by quantitative coronary angiography during intracoronary acetylcholine infusion. Flow cytometry was used to assess endothelial cell apoptosis by quantification of circulating CD31+/annexin V+ apoptotic microparticles in peripheral blood. Increased apoptotic microparticle counts positively correlated with impairment of coronary endothelial function. Multivariate analysis revealed that increased apoptotic microparticle counts predict severe endothelial dysfunction independent of classical risk factors, such as hypertension, hypercholesterolemia, smoking, diabetes, age, or sex. Conclusions—In patients with CAD, endothelial-dependent vasodilatation closely relies on the degree of endothelial cell apoptosis, which is readily measurable by circulating CD31+/annexin V+ apoptotic microparticles. These findings possibly provide new options for risk assessment and may have implications for future treatment strategies of CAD.

Interleukin-6 Induces Oxidative Stress and Endothelial Dysfunction by Overexpression of the Angiotensin II Type 1 Receptor

Abstract— Angiotensin II type 1 (AT1) receptor activation as well as proinflammatory cytokines such as interleukin-6 (IL-6) are involved in the development and progression of atherosclerosis. The detailed underlying mechanisms including interactions between inflammatory agonists and the renin-angiotensin system are poorly understood. Stimulation of cultured rat aortic vascular smooth muscle cells (VSMCs) with IL-6 led to upregulation of AT1 receptor mRNA and protein expression, as assessed by Northern and Western blot experiments. Nuclear run-on and transcription blockade experiments showed that IL-6 increases AT1 receptor mRNA de novo synthesis but not mRNA stability. Preincubation of VSMCs with IL-6 resulted in an enhanced angiotensin II–induced production of reactive oxygen species, as assessed by DCF fluorescence laser microscopy. Treatment of C57BL/6J mice with IL-6 for 18 days increased vascular AT1 receptor expression (real-time RT-PCR) and angiotensin II–induced vasoconstriction, enhanced vascular superoxide production (L-012 chemiluminescence, DHE fluorescence), and impaired endothelium-dependent vasodilatation. These effects were completely omitted in AT1 receptor knockout mice (AT1A−/− mice). Upregulation of vascular AT1 receptor expression in vitro and in vivo is decisively involved in IL-6–induced propagation of oxidative stress and endothelial dysfunction. This interaction of the proinflammatory cytokine IL-6 with the renin-angiotensin system may represent an important pathogenetic mechanism in the atherosclerotic process.

Improvement of Endothelial Function by Systemic Transfusion of Vascular Progenitor Cells

Endothelial dysfunction is characterized by abnormalities in vasoreactivity and is a marker of the extent of atherosclerosis. Cellular repair by circulating progenitor cells of ongoing vascular injury may be essential for vascular integrity and function and may limit abnormalities in vasoreactivity. Apolipoprotein E–deficient (apoE−/−) mice were splenectomized and treated with high-cholesterol diet for 5 weeks, resulting in marked impairment of endothelium-dependent vasodilation of aortic segments as compared with wild-type mice. Intravenous transfusion of 2×107 spleen-derived mononuclear cells (MNCs) isolated from wild-type mice on 3 consecutive days restored endothelium-dependent vasodilation in the apoE−/− mice, as measured 7, 14, and 45 days after transfusion. Histological analyses of aortic tissue identified fluorescent-labeled, exogenously applied progenitor cells that expressed the endothelial cell marker CD31 in the endothelial cell layer of atherosclerotic lesions. Progenitor cell treatment led to increased vascular nitric oxide synthase activity. Transfusion of either in vitro-differentiated Dil-Ac-LDL/lectin-positive endothelial progenitor cells, CD11b-positive (monocyte marker), CD45R-positive (B-cell marker), or Sca-1–positive (stem cell marker) MNC subpopulations significantly improved endothelium-dependent vasodilation, although these treatments were not as effective as transfusion of total MNCs. Depletion of MNCs of either CD11b-positive, CD45R-positive, or Sca-1–positive cells resulted in significant attenuation of endothelium-dependent vasodilation as compared with nondepleted MNCs; however, vasoreactivity was still significantly improved as compared with saline-treated apoE−/− mice. Intravenous transfusion of spleen-derived MNCs improves endothelium-dependent vasodilation in atherosclerotic apoE−/− mice, indicating an important role of circulating progenitor cells for the repair of ongoing vascular injury. More than 1 subpopulation of the MNC fraction seems to be involved in this effect.

Rapid Effects on Vascular Function After Initiation and Withdrawal of Atorvastatin in Healthy, Normocholesterolemic Men

The study was performed on 8 healthy, male sub- jects aged 27 to 31 years who were nonsmokers; body mass index was 20 to 22 and all subjects had a negative family history for vascular disease. Clinical examination and laboratory testing excluded systemic hypertension, hypercholesterolemia (pretreatment to- tal cholesterol 171 15 mg/dl, low- density lipopro- tein 95 7.2 mg/dl, high-density lipoprotein 62 11 mg/dl), diabetes, liver or kidney disorders, and any sign of a recent or ongoing infection. All subjects gave written consent and took 80 mg of atorvastatin be- tween 6:00 and 8.00 P.M. daily. Measurements of fasting serum lipid profiles, liver enzymes, and crea- tine kinase were obtained by standard assays. A broad experimental base suggests that the beneficial effects of statins are mediated through increased bioavailabil- ity of nitric oxide leading to improved endothelial function, 6 and via anti-inflammatory properties. 7 Both the inflammation state and the prognosis of athero- sclerotic disease are evidenced by serum levels of inflammatory proteins such as interleukin-1 or high-

Inhibition of Diet-Induced Atherosclerosis and Endothelial Dysfunction in Apolipoprotein E/Angiotensin II Type 1A Receptor Double-Knockout Mice

Background—Angiotensin II type 1 (AT1) receptor activation is potentially involved in the multifactorial pathogenesis of atherosclerosis. Methods and Results—Apolipoprotein E–deficient (ApoE−/−) mice were crossed with AT1A receptor–deficient (AT1−/−) mice to obtain homozygous double-knockout animals (ApoE−/−-AT1−/− mice). Wild-type (C57BL/6J), ApoE−/−, AT1−/−, and ApoE−/−-AT1−/− mice were fed a high-cholesterol diet for 7 weeks. In contrast to wild-type and AT1−/− mice, this treatment led to severe atherosclerotic lesion formation in the aortic sinus and the aorta (oil red O staining) and to an impaired endothelium-dependent vasodilation (organ chamber experiments with isolated aortic segments) in ApoE−/− mice. In the age-matched ApoE−/−-AT1−/− littermates, development of diet-induced endothelial dysfunction and atherosclerotic lesion formation was profoundly inhibited. Concomitantly, aortic release of superoxide radicals was increased 2-fold in ApoE−/− mice compared with wild-type animals, whereas aortic superoxide production was normalized in ApoE−/−-AT1−/− mice (L-012 chemiluminescence). There were no significant differences in plasma cholesterol levels between ApoE−/− and ApoE−/−-AT1−/− animals. Systolic blood pressure was significantly lower in ApoE−/−-AT1−/− animals than in ApoE−/− mice (tail-cuff measurements). Oral treatment of ApoE−/− mice with either hydralazine or irbesartan reduced systolic blood pressure to the same level; however, only AT1 receptor antagonist treatment reduced atherosclerotic lesion formation and improved endothelial function. Conclusions—Genetic disruption of the AT1A receptor leads to inhibition of vascular oxidative stress, endothelial dysfunction, and atherosclerotic lesion formation in ApoE−/− mice irrespective of blood pressure and plasma cholesterol levels. These results indicate a fundamental role of AT1 receptor activation in atherogenesis.

Rapid Effect of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibition on Coronary Endothelial Function

Abstract— Treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases cardiovascular event rates in hypercholesterolemic patients. Whether statins exert effects within 24 hours on the coronary vasculature in patients with endothelial dysfunction has not been elucidated. Twenty-seven patients with stable angina pectoris and average low-density lipoprotein cholesterol concentrations of 138±9 mg/dL at baseline were allocated to treatment with placebo (14 patients) or 40 mg/d pravastatin (13 patients) in a randomized, double-blind, prospective trial. Coronary endothelial function was assessed before and 24 hours after single treatment by quantitative coronary angiography during intracoronary infusion of nitroglycerin or increasing concentrations of acetylcholine (0.01, 0.1, and 1 &mgr;mol/L). Coronary blood flow reserve was measured by Doppler velocimetry during adenosine infusion. Intracoronary acetylcholine infusion induced abnormal vasoconstriction in both groups before treatment, indicating coronary endothelial dysfunction. Treatment with a single oral 40-mg dose of pravastatin significantly attenuated acetylcholine-mediated vasoconstriction after 24 hours (mean±SE decrease in luminal diameter before and after treatment: 0.01 &mgr;mol/L, 6.1±2.2% versus 3.0±1.2%; 0.1 &mgr;mol/L, 15.6±2.6% versus 7.4±1.8%; P <0.05; 1 &mgr;mol/L, 22.9±2.9% versus 13.2±2.6%; P <0.05). There was no significant difference in the response to acetylcholine in the placebo group (8.1±2.4% versus 9.7±2.4%, 16.1±2.9% versus 16.8±3.2%, and 21.4±3.9% versus 23.3±4.2%). The response to nitroglycerin infusion was not altered in both groups. Increase in coronary blood flow in response to adenosine and coronary flow reserve remained unchanged during placebo and statin treatment. Serum concentrations of blood lipids and high-sensitive C-reactive protein were not significantly altered after 24 hours in response to placebo or pravastatin therapy. Statin treatment improves endothelium-dependent coronary vasomotion within 24 hours in the absence of significant cholesterol reduction. The full text of this article is available online at http://www.circresaha.org.

Angiotensin II Type 1 Receptor Antagonism Improves Hypercholesterolemia-Associated Endothelial Dysfunction

ObjectiveObjective—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-&agr; 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.