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Since the publication of the last American Heart Association scientific statement on air pollution and cardiovascular disease in 2010, unequivocal evidence of the causal role of fine particulate matter air pollution (PM 2.5 , or particulate matter ≤2.5 μm in diameter) in cardiovascular disease has emerged. There is a compelling case to provide the public with practical personalized approaches to reduce the health effects of PM 2.5 . Such interventions would be applicable not only to individuals in heavily polluted countries, high-risk or susceptible individuals living in cleaner environments, and microenvironments with higher pollution exposures, but also to those traveling to locations with high levels of PM 2.5 . The overarching motivation for this document is to summarize the current evidence supporting personal-level strategies to prevent the adverse cardiovascular effects of PM 2.5 , guide the use of the most proven/viable approaches, obviate the use of ineffective measures, and avoid unwarranted interventions. The significance of this statement relates not only to the global importance of PM 2.5 , but also to its focus on the most tested interventions and viable approaches directed at particulate matter air pollution. The writing group sought to provide expert consensus opinions on personal-level measures recognizing the current uncertainty and limited evidence base for many interventions. In doing so, the writing group acknowledges that its intent is to assist other agencies charged with protecting public health, without minimizing the personal choice considerations of an individual who may decide to use these interventions in the face of ongoing air pollution exposure.

Intraplaque hemorrhage promotes atherosclerosis progression, and erythrocytes may contribute to this process. In this study we examined the effects of red blood cells on smooth muscle cell mineralization and vascular calcification and the possible mechanisms involved.Erythrocytes were isolated from human and murine whole blood. Intact and lysed erythrocytes and their membrane fraction or specific erythrocyte components were examined in vitro using diverse calcification assays, ex vivo by using the murine aortic ring calcification model, and in vivo after murine erythrocyte membrane injection into neointimal lesions of hypercholesterolemic apolipoprotein E-deficient mice. Vascular tissues (aortic valves, atherosclerotic carotid artery specimens, abdominal aortic aneurysms) were obtained from patients undergoing surgery.The membrane fraction of lysed, but not intact human erythrocytes promoted mineralization of human arterial smooth muscle cells in culture, as shown by Alizarin red and van Kossa stain and increased alkaline phosphatase activity, and by increased expression of osteoblast-specific transcription factors (eg, runt-related transcription factor 2, osterix) and differentiation markers (eg, osteopontin, osteocalcin, and osterix). Erythrocyte membranes dose-dependently enhanced calcification in murine aortic rings, and extravasated CD235a-positive erythrocytes or Perl iron-positive signals colocalized with calcified areas or osteoblast-like cells in human vascular lesions. Mechanistically, the osteoinductive activity of lysed erythrocytes was localized to their membrane fraction, did not involve membrane lipids, heme, or iron, and was enhanced after removal of the nitric oxide (NO) scavenger hemoglobin. Lysed erythrocyte membranes enhanced calcification to a similar extent as the NO donor diethylenetriamine-NO, and their osteoinductive effects could be further augmented by arginase-1 inhibition (indirectly increasing NO bioavailability). However, the osteoinductive effects of erythrocyte membranes were reduced in human arterial smooth muscle cells treated with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide or following inhibition of NO synthase or the NO receptor soluble guanylate cyclase. Erythrocytes isolated from endothelial NO synthase-deficient mice exhibited a reduced potency to promote calcification in the aortic ring assay and after injection into murine vascular lesions.Our findings in cells, genetically modified mice, and human vascular specimens suggest that intraplaque hemorrhage with erythrocyte extravasation and lysis promotes osteoblastic differentiation of smooth muscle cells and vascular lesion calcification, and also support a role for erythrocyte-derived NO.

Background— Angiotensin II (ATII), a potent vasoconstrictor, causes hypertension, promotes infiltration of myelomonocytic cells into the vessel wall, and stimulates both vascular and inflammatory cell NADPH oxidases. The predominant source of reactive oxygen species, eg, vascular (endothelial, smooth muscle, adventitial) versus phagocytic NADPH oxidase, and the role of myelomonocytic cells in mediating arterial hypertension have not been defined yet. Methods and Results— Angiotensin II (1 mg · kg −1 · d −1 for 7 days) increased the number of both CD11b + Gr-1 low F4/80 + macrophages and CD11b + Gr-1 high F4/80 − neutrophils in mouse aorta (verified by flow cytometry). Selective ablation of lysozyme M-positive (LysM + ) myelomonocytic cells by low-dose diphtheria toxin in mice with inducible expression of the diphtheria toxin receptor (LysM iDTR mice) reduced the number of monocytes in the circulation and limited ATII-induced infiltration of these cells into the vascular wall, whereas the number of neutrophils was not reduced. Depletion of LysM + cells attenuated ATII-induced blood pressure increase (measured by radiotelemetry) and vascular endothelial and smooth muscle dysfunction (assessed by aortic ring relaxation studies) and reduced vascular superoxide formation (measured by chemiluminescence, cytochrome c assay, and oxidative fluorescence microtopography) and the expression of NADPH oxidase subunits gp91 phox and p67 phox (assessed by Western blot and mRNA reverse-transcription polymerase chain reaction). Adoptive transfer of wild-type CD11b + Gr-1 + monocytes into depleted LysM iDTR mice reestablished ATII-induced vascular dysfunction, oxidative stress, and arterial hypertension, whereas transfer of CD11b + Gr-1 + neutrophils or monocytes from gp91 phox or ATII receptor type 1 knockout mice did not. Conclusions— Infiltrating monocytes with a proinflammatory phenotype and macrophages rather than neutrophils appear to be essential for ATII-induced vascular dysfunction and arterial hypertension.

Background— Oxidative injury and dysfunction of the vascular endothelium are early and causal features of many vascular diseases. Single antioxidant strategies to prevent vascular injury have met with mixed results. Methods and Results— Here, we report that induction of a metabolic stress response with adenosine monophosphate kinase (AMPK) prevents oxidative endothelial cell injury. This response is characterized by stabilization of the mitochondrion and increased mitochondrial biogenesis, resulting in attenuation of oxidative c-Jun N-terminal kinase (JNK) activation. We report that peroxisome proliferator coactivator 1α is a key downstream target of AMPK that is both necessary and sufficient for the metabolic stress response and JNK attenuation. Moreover, induction of the metabolic stress response in vivo attenuates reactive oxygen species–mediated JNK activation and endothelial dysfunction in response to angiotensin II in wild-type mice but not in animals lacking either the endothelial isoform of AMPK or peroxisome proliferator coactivator 1α. Conclusion— These data highlight AMPK and peroxisome proliferator coactivator 1α as potential therapeutic targets for the amelioration of endothelial dysfunction and, as a consequence, vascular disease.

Interactions of platelets, leukocytes and the vessel wall play pivotal roles in activating coagulation and precipitating thrombosis. High levels of angiotensin II (ATII) cause arterial hypertension by a complex inflammatory pathway requiring leukocyte recruitment and reactive oxygen species production within the vessel wall coupled to vascular dysfunction. Here we report an upregulation of tissue factor, thrombin-dependent endothelial cell VCAM-1 expression and integrin α4- and platelet-dependent leukocyte adhesion to arterial conductance vessels by ATII in vivo (1mg/kg/d for 7d). ATII-induced vascular dysfunction and Ccl2, VCAM-1 and Ly6C mRNA expression were ameliorated by thrombin inhibition, platelet-depletion as well as in hIL-4R/Ibα mice missing the extracellular ligand binding domains of GPIbα. Blockade of TF during ATII administration also attenuated vascular dysfunction and reduced vascular oxidative stress. Platelet rich plasma (PRP) of ATII-treated mice showed an increased thrombin evoked endogenous thrombin potential (ETP), whereas PRP from mice with pharmacological inhibition of FXI production or of hIL-4R/Ibα mice failed to amplify ETP following chronic ATII exposure. These data show that a FXI-dependent thrombin generation feedback loop requires GPIbα on platelets and suggest that TF-initiated coagulation promotes additional thrombin formation on platelets to cause vascular inflammation. The therapeutic potential of blocking FXI synthesis via antisense oligonucleotides was generalized by experiments with 5/6 nephrectomized (5/6 Nx) rats. 5/6 Nx rats showed after 4 wk of surgery an endothelial dysfunction, increased blood pressure, vascular inflammation and oxidative stress, as well as a elevated ETP compared to sham whereas pharmacological inhibition of FXI synthesis attenuated these effects. Our results reveal a critical role of platelet GPIbα to promote localized thrombin amplification and supporting a FXI-thrombin feedback loop in ATII-induced vascular inflammation. By using a FXI targeted approach, we propose a novel therapeutic possibility to interrupt this heterotypic cellular coagulation-inflammatory circuit.

Background: Pulmonary disease has consistently been related to increased mortality. We investigated central spirometry variables in relation to total mortality in individuals from the general population without diagnosed lung disease also accounting for cardiac function. Methods: In 15,010 individuals from the general population (mean age 55±11 years, age range 35-74 years, 50.5% men) in the Gutenberg Health Study we performed spirometry and multimodal transthoracic echocardiography. The biomarkers N-terminal pro-B-type natriuretic peptide (Nt-proBNP) and high-sensitive troponin I (TnI) were measured in the first 5000 individuals using commercially available assays. Multivariable Cox regression analyses were computed for the lung function parameters forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC) and the Tiffeneau-Pinelli index (FEV1/FVC ratio) after exclusion of individuals with prevalent pulmonary disease (N=1135). Results: The median FEV1/FVC ratio was 79.1%, 19.5% were current smokers, 4.2% had prevalent heart failure. After a median follow-up of 5.5 years 360 deaths were recorded. Multivariable-adjusted Cox regression analyses for the common cardiovascular risk factors (age, sex, body mass index, current smoking, diabetes mellitus, hypertension, dyslipidemia) and heart failure revealed that an increase of one standard deviation (SD) of FEV1 was associated with a 38% risk reduction (hazard ratio (HR) per SD 0.62 [95% confidence interval (CI): 0.52-0.73]). The association remained statistically highly significant after additional adjustment for ejection fraction, stroke volume, Nt-proBNP or TnI. Comparable results were seen for FVC, whereas no association of FEV1/FVC ratio with mortality could be shown. No significant interaction by heart failure was observed. Conclusions: The central lung function parameters FEV1 and FVC were related to all-cause mortality in individuals without manifest lung disease. Our findings indicate that subtle, subclinical pulmonary impairment constitutes a risk for increased mortality independent of cardiac performance.

Despite impressive medical advances1 that have led to diminished cardiovascular death rates in some countries over the past 20 years, cardiovascular disease remains the leading cause of death in developed countries such as the United States. This promises to worsen as a result of aging populations; the incipient obesity and type II diabetes epidemic; sedentary lifestyle; and continued abuse of tobacco, alcohol, and other substances. Cardiovascular disease is clearly multifactorial, and the approach to its prevention necessarily likewise. Candidates for prevention include cyclic guanosine 3′,5′-cyclic monophosphate (cGMP)–dependent signaling networks initiated by natriuretic peptides (NPs) and nitric oxide (NO), which demonstrate characteristics deemed worthy of diagnostic and therapeutic exploitation. cGMP signaling contributes to the function and interaction of several vascular cell types, and its dysfunction could be involved in major destructive processes such as atherosclerosis, hypertension, diabetic complications, (re)stenosis, and tissue infarction, as well as the undermining of clinical therapy like in the case of nitrate tolerance. This review takes a focused look at key elements of the cGMP signaling cascade in vascular tissue, particularly recent advances in our knowledge of cGMP-dependent protein kinase (cGK, also known as PKG) function. Finally, we discuss the potential of clinical monitoring of cGK activity for assessing the functional status of cGMP signaling and for guiding the design of therapeutic strategies to improve vascular function. One of the 2 major synthetic pathways for cGMP generation from guanosine 5′-triphosphate (GTP) is directed by NPs (Figure 1), consisting of atrial (ANP), B-type (BNP), and C-type (CNP) natriuretic peptides, which act via the membrane receptor guanylate cyclases GC-A (highest affinity for ANP, BNP) and GC-B (highest affinity for CNP). ANP and BNP, released from the heart by mechanical stretch in response to increased atrial pressure/volume, and CNP, released from the endothelium, can all cause smooth muscle (SM) …

Background— Polymorphonuclear neutrophils (PMNs) have gained attention as critical mediators of acute coronary syndromes (ACS). Myeloperoxidase (MPO), a hemoprotein abundantly expressed by PMNs and secreted during activation, possesses potent proinflammatory properties and may contribute directly to tissue injury. However, whether MPO also provides prognostic information in patients with ACS remains unknown. Methods and Results— MPO serum levels were assessed in 1090 patients with ACS. We recorded death and myocardial infarctions during 6 months of follow-up. MPO levels did not correlate with troponin T, soluble CD40 ligand, or C-reactive protein levels or with ST-segment changes. However, patients with elevated MPO levels (>350 μg/L; 31.3%) experienced a markedly increased cardiac risk (adjusted hazard ratio [HR] 2.25 [1.32 to 3.82]; P =0.003). In particular, MPO serum levels identified patients at risk who had troponin T levels below 0.01 μg/L (adjusted HR 7.48 [95% CI 1.98 to 28.29]; P =0.001). In a multivariate model that included other biochemical markers, troponin T (HR 1.99; P =0.023), C-reactive protein (1.25; P =0.044), vascular endothelial growth factor (HR 1.87; P =0.041), soluble CD40 ligand (HR 2.78; P P =0.008) were all independent predictors of the patient’s 6-month outcome. Conclusions— In patients with ACS, MPO serum levels powerfully predict an increased risk for subsequent cardiovascular events and extend the prognostic information gained from traditional biochemical markers. Given its proinflammatory properties, MPO may serve as both a marker and mediator of vascular inflammation and further points toward the significance of PMN activation in the pathophysiology of ACS.

3-nitrotyrosine formation is an oxidative protein modification that was first discovered in vivo in the early 1990s by Beckman and colleagues.1,2 The biological relevance of this process was extensively investigated in the subsequent years and further facilitated by the development of 3-nitrotyrosine–specific antibodies.3 Protein tyrosine nitration is mainly mediated by 3 biochemical processes (Figure): (1) by peroxynitrite (ONOO−) formation,4–6 the reaction product of nitric oxide (•NO) and superoxide (•O2−); (2) by a (myelo)peroxidase-catalyzed nitrogen dioxide radical (•NO2) formation from hydrogen peroxide and nitrite;7,8 and (3) by a nonspecific formation of the nitrogen dioxide radical from nitric oxide in oxygenated buffers (reflecting rather artificial ex vivo conditions). Figure. Scheme illustrating antibody (IgG) formation against 3-nitrotyrosine-positive proteins and their involvement in the initiation and propagation of coronary artery disease (CAD). Under physiological conditions, the vascular integrity is mainly guaranteed by nitric oxide (•NO) and prostacyclin (PGI2), causing vasodilation, antiaggregation, anti-inflammation, and intact endothelial barrier function. In the presence of cardiovascular risk factors such as hypertension, chronic smoking, hypercholesterolemia, and diabetes mellitus, there is increased formation of superoxide (•O2−) or increased myeloperoxidase (MPO) activity, with the consequence that •NO is consumed to form peroxynitrite (ONOO−) or nitrogen dioxide radical (•NO2). Peroxynitrite or nitrogen dioxide radical in turn cause tyrosine nitration of prostacyclin synthase (PGIS), with the consequence of reduced prostacyclin formation (PGI2) but also enhanced formation of the vasoconstrictor …

Background — In experimental animal models, long-term in vivo treatment with nitroglycerin (NTG) induces both endothelial dysfunction and tolerance to nitrates. However, it is still controversial whether nitrate tolerance in humans is associated with both endothelial dysfunction and impaired vascular response to nitrovasodilator-derived NO. Methods and Results — Patients undergoing elective bypass surgery were randomized to receive 48 hours of continuous NTG infusion (NTG group) or no nitrate therapy (control group). Segments of surgically removed arteria mammaria, vena saphena, and arteria radialis not required for the bypass procedure were used to examine (1) the vascular responsiveness to NTG and the endothelium-dependent vasodilator acetylcholine; (2) the expression of the NO target, the soluble guanylyl cyclase; (3) the expression of the soluble guanylyl cyclase/cGMP effector target, the cGMP-dependent protein kinase (cGK); and (4) the cGK activity as assessed by the phosphorylation state of its vascular substrate, the vasodilator-stimulated phosphoprotein at serine 239 (P-VASP). NTG treatment caused a marked degree of nitrate tolerance in all 3 vessel types studied and a significant cross-tolerance to the endothelium-dependent vasodilator acetylcholine in A. mammaria and A. radialis. Although soluble guanylyl cyclase, cGK-I, and VASP expression levels were not modified by NTG treatment, a marked decrease of P-VASP, a surrogate parameter for in-vivo cGK-I activity, was observed. Conclusions — We conclude that long-term NTG treatment induces endothelial dysfunction and impaired vascular NO/cGMP signaling in humans, which can be monitored by measuring P-VASP levels.

About a decade ago, the prevailing wisdom was that conventional risk factors explained only approximately half of the risk for a myocardial infarction or stroke. Consequently, efforts to identify novel risk factors were undertaken to improve cardiovascular risk prediction. The hypothesis that inflammation is a central contributor to atherothrombosis has stimulated sustained efforts to characterize the specific molecules and pathways that may be involved and to identify biomarkers in humans that enable detection of underlying inflammatory activation to improve cardiovascular risk prediction. Ridker et al1 pioneered this work and reported that systemic low-grade inflammation assessed by measurements of the acute-phase reactant C-reactive protein is associated with future cardiovascular events in apparently healthy individuals. After this, B-type natriuretic peptide and its N-terminal fragment were highlighted to predict cardiovascular risk even in the general population.2 Overall, C-reactive protein and the N-terminal fragment of B-type natriuretic peptide have most consistently improved risk estimates in specific populations. However, prediction metrics, which inform us about the predictive values beyond classic risk factors, have been less persuasive, and controversial data have been discussed. Recently, a new player entered the field of cardiovascular risk prediction. Troponin, assayed with a highly sensitive test that allows for reliable detection at very low concentrations, is associated with incident heart failure and mortality in the community.3,4 In this issue of Circulation , Saunders et al5 report findings from the Atherosclerosis Risk in Communities (ARIC) Study extending this knowledge and indicating that troponin T concentrations are associated with incident coronary heart disease, mortality, and hospitalization for heart failure in the general population not formerly known to have cardiac disease. Even in the very-low-concentration range, troponin T is related to incident heart failure and mortality. Article see p 1367 The advent of troponin testing in the emergency …

Background: Angiotensin II (ATII) causes hypertension and promotes infiltration of inflammatory cells into the vessel wall. Vascular superoxide formation and shear stress mediated inflammatory remodeling of conduit arteries was shown to be myeloid differentiation factor 88 (MyD88) dependent, but the exact mechanism are unknown. Objective: The goal of this study was to determine the mechanism, how MyD88 contributes to the development of ATII-induced vascular dysfunction and arterial hypertension. Methods and Results: MyD88 deficiency profoundly attenuated ATII-induced (1 mg/kg/d for 7 days) blood pressure increase (measured by radio telemetry) and vascular dysfunction (assessed by aortic ring relaxation studies). Additionally vascular superoxide levels as well as mRNA expression levels of VCAM-1, iNOS, Nox2 were decreased in ATII-infused MyD88 -/- mice compared to WT controls. Aortic flow cytometric analysis revealed that ATII-induced infiltration with CD11b + Ly6C high inflammatory monocytes was significantly dampened in MyD88 -/- mice. ATII led to an increased expression of inflammatory monocyte markers in blood and aorta, which was blocked in MyD88 -/- mice, indicating a role of MyD88 for myeloid cell differentiation. Additionally less IFN-gamma + NK cells were detected in the vessel wall of ATII-treated MyD88 -/- mice and aortic lysates showed reduced mRNA levels of several proinflammatory cytokines like IL-12 and IL-1beta. Bone marrow transfer experiments further revealed a protective effect of MyD88 deficiency in inflammatory cells represented by a reduction of vascular dysfunction and inflammation. Conclusion: We provide first evidence that MyD88 expressed by bone marrow-derived cells plays an essential role in ATII-induced vascular dysfunction and arterial hypertension. Our data indicate that MyD88 is important for cytokine production and might be required for ATII-induced differentiation of monocytes into an inflammatory phenotype.

The recently published Heart Outcomes Prevention Evaluation (HOPE) study,1,2 as well as the Study to Evaluate Carotid Ultrasound Changes in Patients Treated With Ramipril and Vitamin E (SECURE),3 demonstrated that treatment with an ACE inhibitor but not vitamin E had beneficial effects on the prognosis and progression of atherosclerosis. In these studies, long-term ACE inhibition significantly reduced the rate of death, myocardial infarction, and stroke and reduced the intima-to-media ratio of carotid arteries in patients at high risk for cardiovascular events. It is likely that these findings will have wide-ranging implications for the treatment of cardiovascular disease, and the contrasting results with respect to ACE inhibitors and vitamin E provide an opportunity to revisit the role of oxidative stress in vascular disease. ### LDL Oxidation and Cardiovascular Disease A surprising finding of these studies was the ineffectiveness of vitamin E in preventing the clinical manifestations of coronary artery disease and the progression of atherosclerosis. A wealth of previous experimental and epidemiological data suggested that excess LDL oxidation was, in part, responsible for the development of atherosclerosis.4 Because vitamin E can inhibit LDL oxidation ex vivo, a logical strategy to reduce oxidative stress would include the administration of antioxidants such as vitamin E. This approach carries considerable risk if the antioxidant is not active against all oxidants relevant to atherosclerosis. Although vitamin E effectively scavenges lipid peroxyl radicals, it has limited activity against other oxidants such as superoxide, peroxynitrite, and hypochlorous acid that have been implicated in atherosclerosis. Another risk with an antioxidant strategy relates to putative cellular compartments or “microdomains” in the vascular wall that do not contain appreciable amounts of the antioxidant. The sum total of these effects would be continued oxidation even in the presence of the antioxidant. Experimental evidence from both animals and patients suggests that lipid peroxidation does proceed …

Background —We have previously shown that nitroglycerin (NTG) therapy increases vascular expression of endothelin 1 (ET-1) and stimulates vascular superoxide (O 2 ·− ) production via activation of NADH/NADPH oxidases. Both phenomena are stimulated by angiotensin II in vitro, and the renin-angiotensin system is activated during early nitrate therapy. We hypothesized that either angiotensin II or ET-1 may increase vascular O 2 ·− production during nitrate therapy. Methods and Results —In New Zealand White rabbits, 3 days of treatment with NTG patches increased plasma renin activity for the entire treatment period. After 24 hours of NTG treatment, angiotensin II type 1 (AT 1 ) receptor expression and vascular ACE activity were significantly decreased. At this time, constrictions to angiotensin I and II were depressed, but there was no loss of NTG vasodilator potency. Within 3 days of continuous NTG treatment, relaxations to NTG were markedly blunted. This was associated with an increase in AT 1 receptor mRNA expression, a return of ACE activity back to baseline, and a marked increase in constrictions to angiotensin I and II despite continuously increased plasma renin activity. Tolerance was associated with a 2-fold increase in vascular O 2 ·− , as estimated by lucigenin-enhanced chemiluminescence. Concomitant treatment with the AT 1 receptor antagonist losartan (5 to 25 mg · kg −1 · d −1 ) dose-dependently normalized vascular O 2 ·− and prevented tolerance to NTG and cross-tolerance to endogenous nitric oxide released by acetylcholine. The nonselective ET-1 receptor blocker bosentan (100 mg · kg −1 · d −1 ) had similar but less pronounced effects. Conclusions —The positive effects of AT 1 and ET-1 receptor blockade on tolerance and O 2 ·− production imply a pathophysiological role for angiotensin II and to some extent for ET-1 in the development of nitrate tolerance.

Background —Angiotensin II activates NAD(P)H-dependent oxidases via AT 1 -receptor stimulation, the most important vascular source of superoxide (O 2 · − ). The AT 1 receptor is upregulated in vitro by low-density lipoprotein. The present study was designed to test whether hypercholesterolemia is associated with increased NAD(P)H-dependent vascular O 2 · − production and whether AT 1 -receptor blockade may inhibit this oxidase and in parallel improve endothelial dysfunction. Methods and Results —Vascular responses were determined by isometric tension studies, and relative rates of vascular O 2 · − production were determined by use of chemiluminescence with lucigenin, a cypridina luciferin analogue, and electron spin resonance studies. AT 1 -receptor mRNA was quantified by Northern analysis, and AT 1 -receptor density was measured by radioligand binding assays. Hypercholesterolemia was associated with impaired endothelium-dependent vasodilation and increased O 2 · − production in intact vessels. In vessel homogenates, we found a significant activation of NADH-driven O 2 · − production in both models of hyperlipidemia. Treatment of cholesterol-fed animals with the AT 1 -receptor antagonist Bay 10-6734 improved endothelial dysfunction, normalized vascular O 2 · − and NADH-oxidase activity, decreased macrophage infiltration, and reduced early plaque formation. In the setting of hypercholesterolemia, the aortic AT 1 receptor mRNA was upregulated to 166±11%, accompanied by a comparable increase in AT 1 -receptor density. Conclusions —Hypercholesterolemia is associated with AT 1 -receptor upregulation, endothelial dysfunction, and increased NADH-dependent vascular O 2 · − production. The improvement of endothelial dysfunction, inhibition of the oxidase, and reduction of early plaque formation by an AT 1 -receptor antagonist suggests a crucial role of angiotensin II–mediated O 2 · − production in the early stage of atherosclerosis.

During the past decade, HMG-CoA reductase inhibitors (statins) have been shown to improve survival in patients with cardiovascular disease. Initially, the beneficial effects of statins were attributed simply to lipid reduction1; however, more recent data suggest that “pleiotropic” properties such as improvement of endothelial dysfunction, increased nitric oxide bioavailability, and antioxidative and antiinflammatory properties may contribute to the improvement of prognosis in patients with coronary artery disease. Many of these pleiotropic effects of statins are mediated by the ability to block the synthesis of important isoprenoid intermediates, which have been shown to serve as lipid attachments for a variety of intracellular signaling molecules. In particular, the inhibition of the small guanosine triphosphate–binding proteins rho, ras, and rac, the membrane localization of which depends on isoprenylation, may play an important role in mediating the direct effects of statins on the vascular wall. Thus, the characteristics of this class of drugs may explain the dramatic effects on the progression of atherosclerosis and therefore on survival in patients with established coronary artery disease.

Background Rebound myocardial ischemia develops in patients with unstable or stable angina pectoris after sudden cessation of nitroglycerin therapy. Long-term nitroglycerin infusion is associated with increases in plasma renin activity and catecholamine release rates, both of which may lead to excess angiotensin II and α-adrenergic–mediated vasoconstriction, particularly on withdrawal of nitroglycerin. Methods and Results Chronically instrumented dogs were treated for 5 days with nitroglycerin (1.5 μg·kg −1 ·min −1 IV) alone or in combination with the angiotensin-converting enzyme (ACE) inhibitor enalapril (0.1 mg/kg two times daily or 1 mg/kg). With long-term nitroglycerin therapy, the left anterior circumflex artery was maximally dilated 4 hours after the start of nitroglycerin infusion (9.5±0.6%) and returned to baseline levels within the third day of treatment (baseline, 2.52±0.07 mm; day 3, 2.55±0.07 mm; P =NS), indicating a complete loss of nitroglycerin-induced coronary vasodilatation. Nitroglycerin infusion also was accompanied by a transient increase in plasma renin activity. Sudden withdrawal of nitroglycerin infusion caused a progressive constriction of the left anterior circumflex artery, which peaked 4 hours after nitroglycerin infusion cessation (−7.8±0.2%). This occurred in the absence of elevated plasma renin activity. Concomitant treatment with high-dose enalapril (1 mg·kg −1 ·d −1 ) markedly reduced the degree of tolerance and prevented the rebound constriction on cessation of nitroglycerin therapy. Conclusions Long-term ACE inhibition with high-dose enalapril reduces nitroglycerin tolerance and prevents rebound vasoconstriction in coronary arteries. These phenomena were not associated with an activated circulating renin-angiotensin system. This observation suggests that during long-term nitroglycerin treatment, intrinsic abnormalities of the vascular smooth muscle may have developed that are suppressed by concomitant ACE inhibitor therapy. The present study also favors a combination of nitroglycerin and ACE inhibitors to maintain nitrate sensitivity of the vasculature during long-term nitroglycerin treatment.

Enzymes are critical components of every cell, and their activity is tightly regulated by substrate availability, competitive or allosteric inhibitors, and the presence of cofactors. Enzymatic activity may also be modulated by posttranslational modifications, such as cleavage of the polypeptide chain or phosphorylation/dephosphorylation of amino acid residues. Under pathological conditions, other modifications, such as oxidation or nitration, may occur, thereby disturbing enzyme activity. Beside these well-known principles of enzyme biology, temperature and pH are crucial determinants of enzyme activity, but corresponding changes in the cellular environment have attracted surprisingly little attention as putative disease mechanisms. Changes in intracellular pH can affect the ionization state of acidic or basic amino acid residues, which may disrupt ionic bonds that help to determine the 3-dimensional shape of the enzyme. These conformational alterations can lead to inactivity of the enzyme, because they may prevent binding of the substrate or cofactors. In addition, the modification of intracellular pH may change the charge properties of the substrate, so that either the substrate cannot bind to the active site or it cannot undergo catalysis.

Although the short-term vasodilatory properties of organic nitrates are potent and well known, a number of vascular and extravascular changes have been shown to compromise their hemodynamic effects on long-term administration. Among these changes, systemic phenomena such as neurohormonal activation and intravascular volume expansion1 as well as specific vascular changes such as increased vascular superoxide (O2·−) production,2 increased sensitivity to vasoconstrictors,3 and decreased responsiveness to nitric oxide (NO) donors4,5 have long been identified as playing a role. Several hypotheses have been proposed to explain these abnormalities, and over the last 15 years, our groups have focused on the concept that an inappropriate production of reactive oxygen species (ROS), an impairment in the scavenging of these mediators (Figure 1), or both might have a crucial mechanistic importance in all these modifications.6,7 Independently of the role of ROS in tolerance, the possibility that nitrate-induced oxidative stress might affect patients' prognosis has important implications, and the observation that long-term therapy with most of the drugs in this class causes endothelial dysfunction, the prognostic significance of which is well accepted in patients with coronary artery disease, hypertension, and heart failure,8 should not be taken as an academic curiosity. Figure 1. Free radical biochemistry. The radical nitric oxide reacts with superoxide to form the highly reactive intermediate peroxynitrite. Superoxide is dismutated by superoxide dismutase (SOD), leading to the formation of hydrogen peroxide (H2O2) and molecular oxygen (O2). Beyond these as-yet insufficiently investigated prognostic implications, recognition of the role of ROS suggests that a number of interventions thought to interfere with the vascular redox balance might also retard or prevent the development of nitrate tolerance and nitrate-induced side effects. Evidence that therapy with angiotensin-converting enzyme (ACE) inhibitors, angiotensin-1 receptor blockers, certain …

BACKGROUND L-Methionine potentiates systemic hemodynamic effects of intravenous glyceryl trinitrate (GTN) in tolerant and nontolerant patients to a similar extent as N-acetylcysteine (NAC). This potentiation of GTN action by L-methionine has been attributed to enhanced intracellular formation of nitrosothiols, known to be potent stimulators of soluble guanylyl cyclase. This study was performed to analyze directly the effects of L-methionine on GTN-induced dilation of large epicardial arteries and the venous capacitance system of the dog in the tolerant and nontolerant states. Cultured rat aortic vascular smooth muscle cells and purified guanylyl cyclase were used to study potential intracellular and extracellular mechanisms responsible for this interaction. METHODS AND RESULTS In awake nontolerant dogs, L-methionine (100 mg/kg) potentiated the tachycardic response to GTN (5.0 and 15 micrograms/kg/min) and enhanced the hypotensive action of GTN (1.5 and 5.0 micrograms/kg/min) in anesthetized, nonreflexic dogs. In nontolerant and tolerant dogs, however, L-methionine did not alter the dose-response of large epicardial artery dilation to intravenous GTN challenges and did not modify nitrate tolerance of the low pressure system of the dog. The infusion of L-methionine (100 mg/kg) significantly increased plasma methionine levels (from 52 +/- 12 to 1,141 +/- 239 microM), cystine levels (from 12 +/- 4 to 26 +/- 7 microM), but not homocystine levels. In vitro, the L-methionine conversion product L-cysteine (0.1-1.0 mM) but not homocysteine significantly enhanced the augmentation of purified guanylyl cyclase activity by GTN (100 microM). Incubation of cultured rat aortic smooth muscle cells with L-methionine (10 microM or 1 mM) did not result in a significant increase of free intracellular sulfhydryl group content. CONCLUSIONS The L-methionine conversion product L-cysteine mediates tolerance independent the potentiation of GTN action. This may result from an L-cysteine-induced formation of a vasoactive metabolite of GTN (nitric oxide) or nitrosothiol. This effect occurs primarily in the resistance vessel circulation, not in large epicardial arteries and veins. The lack of effect of L-methionine on sulfhydryl group content in large conductance vessels indicates that hepatic L-methionine metabolism constitutes the significant source of L-cysteine. These findings strongly suggest that administration of sulfhydryl-group precursor L-methionine does not represent a therapeutic alternative to a nitrate-free interval to restore nitrate sensitivity in tolerant large epicardial arteries and veins.

BACKGROUND The present study was designed to define the prevalence and characteristics of skeletal muscle alterations in patients with chronic heart failure (CHF) and their relation to exercise capacity. METHODS AND RESULTS The ultrastructure of skeletal muscle was analyzed by ultrastructural morphometry in 57 patients with CHF and 18 healthy controls. The volume density of mitochondria (Vvm) and the surface density (Svmc) of mitochondrial cristae were evaluated as a structural correlate of oxidative capacity of skeletal muscle. Vvm and Svmc were reduced by approximately 20% in patients with severe CHF irrespective of age and etiology. The cytochrome oxidase activity in mitochondria as determined by cytochemistry and subsequent morphometry in a subset of patients (n = 10) was significantly decreased in heart failure (p less than 0.01). The capillary length density of skeletal muscle was reduced in CHF (n = 12, p less than 0.05), and the fiber type distribution was shifted to type II fibers (n = 15, p less than 0.05). Vvm and Svmc were significantly related to peak exercise VO2 (r = 0.56, p less than 0.001, n = 60) and to VO2 at anaerobic threshold (r = 0.535, p less than 0.0001, n = 60). In 16 patients with severe heart failure, Vvm was inversely related to the duration of heart failure (r = 0.545, p less than 0.03). In 11 patients who underwent repeat biopsies after 4 months, a correlation was observed between the change in Vvm and the change in peak exercise VO2 (r = 0.89, p less than 0.001). CONCLUSIONS These findings indicate that patients with CHF develop significant ultrastructural abnormalities of skeletal muscle reflecting a depressed oxidative capacity of working muscle. It appears that these alterations of skeletal muscle contribute to the decreased exercise capacity of these patients but are, in principle, reversible by an effective treatment regimen.

We thank Jaquinandi et al for their interest in our publication. We want to stress that the patient population of the AtheroGene study is not comparable with the patient populations reported in the articles from Lee et al and Kreitner et al, which included only patients with known advanced peripheral arterial disease.1,2 The AtheroGene study includes primarily patients with coronary artery disease, no patient had rest pain or peripheral ulcers and only 52 patients (6.3%) had intermittent claudication.3 In addition, …

We examined the mechanisms involved in the cardiovascular and renal response to prolonged infusion of atrial natriuretic factor (ANF) in patients with chronic heart failure. ANF infusion was titrated to produce a 30% decrease in pulmonary capillary wedge pressure or a 20% increase in cardiac output, and this dose (average, 75 +/- 4 ng/kg/min) was then administered for 20 hours. The short-term response to ANF included significant reductions in central filling pressures, increases in cardiac output, modest increases in diuresis and glomerular filtration rates, significant reduction in plasma aldosterone levels, and a 3.6-fold increase in plasma cyclic GMP levels. During prolonged infusion, plasma cGMP levels and cardiac output gradually returned to baseline. Similarly, the initially increased diuretic effects were completely abolished during prolonged ANF infusion, although plasma alpha-hANF levels remained consistently elevated above baseline values (control, 198 +/- 38; titration, 2,760 +/- 596; 20 hours, 3,499 +/- 659 pg/ml). Four hours after beginning the ANF infusion, marked increases in hematocrit levels were noted (42.5 +/- 1.0% versus 45.3 +/- 1.4%, control and infusion, respectively, p less than 0.05); during this time, no change in total plasma protein concentration occurred, indicating extravascular shift of fluid and plasma proteins. No evidence was noted for activation of vasoconstrictor hormones during prolonged ANF infusion, although mean arterial pressure was significantly reduced throughout the infusion period. Plasma pro-ANF (31-67) levels, determined as a marker for endogenous ANF secretion, were significantly suppressed as were the reductions of central filling pressures. After ANF discontinuation, heart rate and pulmonary capillary wedge pressure increased significantly above baseline values without evidence for sympathetic stimulation. We conclude that 1) prolonged infusion of ANF causes only transient increases in plasma cGMP levels but a sustained reduction of the cardiac release of ANF and that 2) the beneficial hemodynamic effects of ANF, that is, unloading of the ventricles, may be associated with or, in part, may be secondary to a shift of plasma constituents into the extravascular space. The latter may limit the therapeutic potential of ANF for long-term treatment.

Increased peripheral vascular resistance is a hallmark of advanced chronic congestive heart failure (CHF) and contributes to the phenomenon of “increased afterload” that complicates this condition. Multiple factors probably underlie this phenomenon, including increased water and sodium content of the vasculature, increased neurohormonal activation, and intrinsic abnormalities of the vasculature.1 During the past decade, it has also been shown that endothelium-dependent vasodilation is strikingly abnormal in both experimental animals and humans with compromised cardiac function.2 3 Given that endothelial regulation of vasomotion plays a major role in the control of systemic hemodynamics, this phenomenon is probably a major cause of increased systemic vascular resistance and afterload in heart failure. Because endothelial regulation of vascular tone is mediated predominantly by endothelium-derived nitric oxide (NO), numerous studies have examined abnormalities of the l-arginine/NO pathway in heart failure. Two major abnormalities have surfaced as important. Several studies have suggested that CHF leads to a decline in expression of endothelial cell NO synthase (eNOS), which is ultimately responsible for endothelial production of NO. Wang et al4 produced heart failure in dogs by cardiac pacing at a rapid rate for 4 weeks. Microvascular endothelial cells released markedly less nitrite (the stable degradation product of NO) than cells from hearts of normal animals. Using the same heart failure model, Smith and colleagues5 observed a decrease in the expression of both eNOS and cyclooxygenase 1, the enzyme responsible for production of prostacyclin. CHF is also associated with increased circulating levels of the cytokine TNF-α.6 In vitro, TNF-α increases degradation of eNOS mRNA, most likely via …

Circulating levels of the TGF β-related cytokine, growth-differentiation factor-15 (GDF-15), provide independent prognostic information in patients with unstable coronary artery disease (CAD). To explore the prognostic utility of GDF-15 in patients with stable CAD, we analyzed the relation of GDF-15 to mortality and cardiovascular (CV) events in the AtheroGene registry which enrolled consecutive patients with stable angina and at least one stenosis >30% in a larger coronary artery. Patients were followed for a median of 3.6 years. Serum samples for measurement of GDF-15 along with other biomarkers were available from 1352 patients. Two pre-specified cutoff points (1200 and 1800 ng/L) were used to identify different risk groups. 55.9%, 26.4%, and 17.7% of the patients presented with GDF-15 values 1800 ng/L, respectively. Increasing levels of GDF-15 were related to age (P

Purpose: Chronic nitrate therapy remains controversial in patients with coronary artery disease (CAD) due to induction of tolerance and deterioration of endothelial dysfunction. Pentaerithri-tyltetranitrate (PETN) differs from other organic nitrates by the lack of tolerance induction and by antioxidative properties. We hypothesized that treatment with PETN improves endothelial dysfunction in patients with CAD. Methods: In a prospective, double-blind trial, we randomly assigned 80 patients to double-blind treatment for 8 weeks with oral PETN 80mg t.i.d. (PETN) or placebo (C), respectively. The primary endpoint was the absolute change in flow-mediated dilation of the brachial artery (FMD) from baseline to follow-up. Secondary endpoints were the changes in nitroglycerin-mediated dilation (NMD), peripheral arterial tonometry (EndoPAT2000) index as a marker of endothelial function of small arteries, the augmentation index (AI) as a marker of arterial stiffness and the activity of the PETN bioactivating enzyme aldehydedehydrogenase-2 (ALDH-2) in monocytes from baseline to follow-up. Raw data entry, data monitoring and statistic analysis were independently performed by the coordination centre of clinical trials (KKS), Mainz. The trial was registered as ISRCTN 14741768. Results: Both treatment groups were comparable regarding age, gender, distribution of cardiovascular risk factors and concomitant medication. There was no difference in the mean (±SD) change in FMD between the two treatment groups (PETN: +1.6±3.3% vs. C: +1.4±4.1% p=0.7). NMD significantly rose after treatment with PETN compared to C (PETN: +3.8±5.5% vs. C: +0.6±4.2% p=0.0036). Mean PAT-Index, AI and ALDH-2 activity remained unchanged in both treatment groups. Conclusions: The three main findings of our trial are: In contrast to other organic nitrates PETN does not deteriorate endothelial dysfunction (1), does not impair ALDH-2 activity (2) and does not induce cross tolerance to nitroglycerin (3). We conclude that chronic PETN therapy in patients with CAD may be established for symptomatic treatment without adverse effects on endothelial function.

Background— An ankle-brachial index (ABI; ratio of ankle and brachial systolic blood pressure) Methods and Results— In 831 patients admitted with chest pain for diagnostic heart catheterization, blood pressure of both anterior and posterior tibial arteries was measured. ABI was calculated for each leg with the higher of the 2 ankle pressures (current definition of the American Heart Association) or with the lower of the 2 ankle pressures (modified definition) in relation to the higher of the left or right brachial systolic blood pressure. For each patient, the lower ABI from both legs was used for further evaluation. Fifteen patients (1.8%) with ABI >1.5 were excluded. We compared patients with ABI with PAD , n=204 [25.0%]), those with ABI ≥0.9 according to the modified definition ( without PAD , n=524 [64.2%]), and those with ABI suspected PAD , n=88 [10.8%]). Follow-up data (median 6.6 years) were available for 812 patients (99.5%); 157 patients (19.3%) experienced cardiovascular events (cardiovascular death, myocardial infarction, or stroke). Patients without PAD had the lowest cardiovascular event rate, whereas event rates were comparable for patients with PAD and those with suspected PAD (14.8% versus 28.4% versus 25.0%, respectively). In a fully adjusted Cox regression analysis that included patients without PAD as the reference group, the hazard ratio (95% CI) was 1.56 (0.97 to 2.53) for patients with suspected PAD and 1.67 (1.16 to 2.40) for patients with PAD. Conclusions— When the higher ankle pressure is used for ABI calculation, a group of patients at high risk for cardiovascular events is overlooked. With a simple modification of ABI (use of the lower instead of the higher ankle pressure), more patients at risk could be identified.

Background— The use of the Endeavor stent might reduce restenosis and stent thrombosis at 9 months. Methods and Results— Patients (n=1197) treated for single coronary artery stenosis were enrolled in a prospective, randomized, double-blind study and randomly assigned to receive the Endeavor zotarolimus-eluting phosphorylcholine polymer–coated stent (n=598) or the same bare metal stent but without the drug or the polymer coating (n=599). The 2 groups were well matched in baseline characteristics. Diabetes was present in 20.1% of patients; the mean reference vessel diameter was 2.75 mm; and the mean lesion length was 14.2 mm. The primary end point of target vessel failure at 9 months was reduced from 15.1% with the bare metal stent to 7.9% with the Endeavor ( P =0.0001), and the rate of major adverse cardiac events was reduced from 14.4% with the bare metal stent to 7.3% with the Endeavor ( P =0.0001). Target lesion revascularization was 4.6% with Endeavor compared with 11.8% with the bare metal stent ( P =0.0001). The rate of stent thrombosis was 0.5% with the Endeavor, which was not significantly different from 1.2% with the bare metal stent. In 531 patients submitted to angiographic follow-up, late loss was reduced from 1.03±0.58 to 0.61±0.46 ( P P P P Conclusions— Compared with bare metal stents, the Endeavor stent is safe and reduces the rates of clinical and angiographic restenosis at 9, 12, and 24 months.

Nitric oxide (NO·) is an important protective molecule in the vasculature, and endothelial NO· synthase (eNOS) is responsible for most of the vascular NO· produced. A functional eNOS oxidizes its substrate l -arginine to l -citrulline and NO·. This normal function of eNOS requires dimerization of the enzyme, the presence of the substrate l -arginine, and the essential cofactor (6 R )-5,6,7,8-tetrahydro- l -biopterin (BH 4 ), one of the most potent naturally occurring reducing agents. Cardiovascular risk factors such as hypertension, hypercholesterolemia, diabetes mellitus, or chronic smoking stimulate the production of reactive oxygen species in the vascular wall. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases represent major sources of this reactive oxygen species and have been found upregulated and activated in animal models of hypertension, diabetes, and sedentary lifestyle and in patients with cardiovascular risk factors. Superoxide (O 2 · − ) reacts avidly with vascular NO· to form peroxynitrite (ONOO − ). The cofactor BH 4 is highly sensitive to oxidation by ONOO − . Diminished levels of BH 4 promote O 2 · − production by eNOS (referred to as eNOS uncoupling). This transformation of eNOS from a protective enzyme to a contributor to oxidative stress has been observed in several in vitro models, in animal models of cardiovascular diseases, and in patients with cardiovascular risk factors. In many cases, supplementation with BH 4 has been shown to correct eNOS dysfunction in animal models and patients. In addition, folic acid and infusions of vitamin C are able to restore eNOS functionality, most probably by enhancing BH 4 levels as well.

Background— Interleukin (IL)-18 plays a key role in atherosclerosis and its complications. The present study investigated the genetic variability of 4 genes of the IL-18 system— IL18 , IL18R1 , IL18RAP , and IL18BP —in relation to circulating IL-18 levels and cardiovascular mortality. Methods and Results— Twenty-two polymorphisms were genotyped in 1288 patients with coronary artery disease prospectively followed up during a median period of 5.9 years. The end point was death from cardiovascular causes (n=142). Baseline IL-18 levels were predictive of cardiovascular deaths occurring during ≤4 years of follow-up (HR=2.96, 95% CI 1.54 to 5.70, P =0.001 for the top compared with the bottom quartile) but not of later deaths. Haplotypes of the IL18 gene were associated with IL-18 levels ( P =0.002) and cardiovascular mortality ( P =0.006) after adjustment for cardiovascular risk factors. The same haplotype was associated with both a 9% lowering effect on IL-18 levels and a protective effect on risk (HR=0.57, 95% CI 0.36 to 0.92). IL18 haplotypes explained only 2% of IL-18 variability. Adjustment for baseline IL-18 levels abolished the association of haplotypes with cardiovascular risk. The haplotype associated with phenotypes was the only one carrying the minor allele of the IL18 /A+183G polymorphism located in the 3′untranslated region and potentially affecting mRNA stability. The other genes of the system were not related to IL-18 levels or cardiovascular outcome. Conclusion— Variations of the IL18 gene consistently influence circulating levels of IL-18 and clinical outcome in patients with coronary artery disease, which supports the hypothesis of a causal role of IL-18 in atherosclerosis and its complications.

Background— Polymer-controlled paclitaxel-eluting stents have shown a pronounced reduction in neointimal hyperplasia compared with bare metal stents (BMS). The aim of this substudy was to evaluate local arterial responses through the use of serial quantitative intravascular ultrasound (IVUS) analyses in the TAXUS II trial. Methods and Results— TAXUS II was a randomized, double-blind study with 536 patients in 2 consecutive cohorts comparing slow-release (SR; 131 patients) and moderate-release (MR; 135 patients) paclitaxel-eluting stents with BMS (270 patients). This IVUS substudy included patients treated with one study stent who underwent serial IVUS examination after the procedure and at 6-month follow-up (BMS, 152 patients; SR, 81; MR, 81). The analyzed stented segment (15 mm) was divided into 5 subsegments in which mean vessel area (VA), stent area (SA), lumen area (LA), intrastent neointimal hyperplasia area (NIHA), and peristent area (VA−SA) were measured. NIHA was significantly reduced in SR (0.7±0.9 mm 2 , P 2 , P 2 ), with no differences between the two paclitaxel-eluting release formulations. Longitudinal distribution of neointimal hyperplasia throughout the paclitaxel-eluting stent was uniform. Neointimal growth was independent of peristent area at postprocedure examination in all groups. There were progressive increases in peristent area from BMS to SR to MR (0.5±1.7, 1.0±1.8, and 1.4±2.0 mm 2 , respectively; P Conclusions— Both SR and MR paclitaxel-eluting stents prevent neointimal formation to the same degree compared with BMS. However, the difference in peristent remodeling suggests a release-dependent effect between SR and MR.

Background— Platelet glycoprotein IIb/IIIa receptor blockade not only enhances epicardial flow but also improves microvascular perfusion. Inhibition of abnormal platelet–endothelial interactions may contribute to this beneficial effect. The present study was designed to determine whether glycoprotein IIb/IIIa receptor blockade influences endothelial vasomotor function and NO bioactivity in patients with coronary artery disease. Methods and Results— Forty patients with symptomatic coronary artery stenosis were studied before planned percutaneous coronary intervention. By using venous occlusion plethysmography, endothelium-dependent and -independent vasodilation was determined by measuring forearm blood flow responses to acetylcholine with and without N G -monomethyl- l -arginine (L-NMMA) and sodium nitroprusside. Vascular function tests were repeated during glycoprotein IIb/IIIa receptor blockade by tirofiban in 27 patients and by eptifibatide in 13 patients. A subgroup of 10 patients was retested 6 hours after stopping infusion of tirofiban. Glycoprotein IIb/IIIa receptor blockade by both substances improved acetylcholine-induced vasodilation and L-NMMA responses. Six hours after withdrawal of tirofiban infusion, the beneficial effects were not evident. Sodium nitroprusside–induced vasodilation was not changed by glycoprotein IIb/IIIa receptor blockade. Conclusions— These findings support the concept that abnormal platelet-endothelial interactions contribute to endothelial dysfunction and impaired NO bioactivity in patients with symptomatic coronary artery disease.

Nitroglycerin (NTG) has been the foremost anti-ischemic agent used in clinical medicine for more than a century. NTG is a potent vasodilator of veins, arterial conductance vessels, and collaterals that has minimal effects on arteriolar tone. At the cellular level, NTG is biotransformed by a still unknown enzymatic process in endothelial cells, smooth muscle, and to some extent platelets, causing it to release the vasodilator and anti-aggregatory principle nitric oxide. The 2 major drawbacks of nitrate therapy that have been shown to be important are the rapid development of nitrate tolerance1 and endothelial dysfunction2,3⇓ within several days of prolonged NTG treatment. There is a growing body of evidence that both NTG-induced side effects may be at least in part secondary to NTG-stimulated production of oxygen-derived free radicals within vascular tissue.4–6⇓⇓ Increased oxidative stress correlates positively with increased cardiovascular event rates,7 which may, at least in part, explain why NTG-therapy either failed to improve or worsened the prognosis in patients with acute myocardial infarction and chronic ischemic heart disease, respectively. See p 208 For years, investigators have been debating whether NTG has significant antiplatelet activity. Several studies demonstrated that inhibition of platelet aggregation by NTG occurs in suprapharmacological dosis, denying any clinically relevant antiplatelet activity. Whether or not NTG inhibits platelet aggregation clearly depends on the chosen experimental conditions. For example, in washed platelets, the lowest effective concentrations of NTG that inhibit platelet aggregation are consistently in the μmol/L range and are therefore in concentrations that are 1000 times higher than those achieved with in vivo NTG therapy (nmol/L range), a finding which has raised doubt about the physiological significance of platelet inhibition by NTG. As mentioned above, NTG has to be biotransformed in order to release NO. The weak antiplatelet activity of NTG, …

To the Editor: In a recent paper, Tulis and coworkers1 reported that transfectional overexpression of heme oxygenase-1 (HO-1) inhibits arterial remodeling after balloon angioplasty via a mechanism involving apoptosis. HO-1 is a stress response protein that degrades heme to carbon monoxide, biliverdin, and nonheme iron. In general, overexpression of HO-1 plays a cytoprotective role against a variety of oxidative stimuli. Interestingly, cells overexpressing HO-1 exhibit low levels of free iron because of the upregulation of ferritin and the extraction of iron into the extracellular space.2 The elimination of pro-oxidant iron from the cell is considered to be an important mechanism of HO-1–mediated protection against oxidative stress.2 How HO-1 overexpression stimulates apoptosis in the medial wall of injured artery is unclear. We believe that, among potential mechanisms of …

Background Endothelial function is impaired in coronary artery disease and may contribute to its clinical manifestations. Increased oxidative stress has been linked to impaired endothelial function in atherosclerosis and may play a role in the pathogenesis of cardiovascular events. This study was designed to determine whether endothelial dysfunction and vascular oxidative stress have prognostic impact on cardiovascular event rates in patients with coronary artery disease. Methods and Results Endothelium-dependent and -independent vasodilation was determined in 281 patients with documented coronary artery disease by measuring forearm blood flow responses to acetylcholine and sodium nitroprusside using venous occlusion plethysmography. The effect of the coadministration of vitamin C (24 mg/min) was assessed in a subgroup of 179 patients. Cardiovascular events, including death from cardiovascular causes, myocardial infarction, ischemic stroke, coronary angioplasty, and coronary or peripheral bypass operation, were studied during a mean follow-up period of 4.5 years. Patients experiencing cardiovascular events (n=91) had lower vasodilator responses to acetylcholine ( P P P P =0.001), the effect of vitamin C ( P =0.001), and age ( P =0.016) remained independent predictors of cardiovascular events. Conclusions Endothelial dysfunction and increased vascular oxidative stress predict the risk of cardiovascular events in patients with coronary artery disease. These data support the concept that oxidative stress may contribute not only to endothelial dysfunction but also to coronary artery disease activity.

Background The efficacy of nitroglycerin (NTG) as a vasodilator is limited by tolerance, which develops shortly after treatment begins. In vascular smooth muscle cells (VSMCs), NTG is denitrated to form nitric oxide (NO), which activates guanylyl cyclase and generates cGMP. cGMP plays a key role in nitrate-induced vasodilation by reducing intracellular Ca 2+ concentration. Therefore, one possible mechanism for development of nitrate tolerance would be increased activity of the cGMP phosphodiesterase (PDE), which decreases cGMP levels. Methods and Results To test this hypothesis, rats were made tolerant by continuous infusion of NTG for 3 days (10 μg · kg −1 · min −1 SC) with an osmotic pump. Analysis of PDE activities showed an increased function of Ca 2+ /calmodulin (CaM)–stimulated PDE (PDE1A1), which preferentially hydrolyzes cGMP after NTG treatment. Western blot analysis for the Ca 2+ /CaM-stimulated PDE revealed that PDE1A1 was increased 2.3-fold in NTG-tolerant rat aortas. Increased PDE1A1 was due to mRNA upregulation as measured by relative quantitative reverse transcription–polymerase chain reaction. The PDE1-specific inhibitor vinpocetine partially restored the sensitivity of the tolerant vasculature to subsequent NTG exposure. In cultured rat aortic VSMCs, angiotensin II (Ang II) increased PDE1A1 activity, and vinpocetine blocked the effect of Ang II on decrease in cGMP accumulation. Conclusions Induction of PDE1A1 in nitrate-tolerant vessels may be one mechanism by which NO/cGMP-mediated vasodilation is desensitized and Ca 2+ -mediated vasoconstriction is supersensitized. Inhibiting PDE1A1 expression and/or activity could be a novel therapeutic approach to limit nitrate tolerance.

Background —Chronic in vivo treatment with nitroglycerin (NTG) induces tolerance to nitrates and cross-tolerance to nitrovasodilators and endothelium-derived nitric oxide (NO). We previously identified increased vascular superoxide formation and reduced NO bioavailability as one causal mechanism. It is still controversial whether intracellular downstream signaling to nitrovasodilator-derived NO is affected as well. Methods and Results —We therefore studied the effects of 3-day NTG treatment of rats and rabbits on activity and expression of the immediate NO target soluble guanylyl cyclase (sGC) and on the cGMP-activated protein kinase I (cGK-I). Tolerance was induced either by chronic NTG infusion via osmotic minipumps (rats) or by NTG patches (rabbits). Western blot analysis, semiquantitative reverse transcription–polymerase chain reaction, and Northern blot analysis revealed significant and comparable increases in the expression of sGC α 1 and β 1 subunit protein and mRNA. Studies with the oxidative fluorescent dye hydroethidine revealed an increase in superoxide in the endothelium and smooth muscle. Stimulation with NADH increased superoxide signals in both layers. Although cGK-I expression in response to low-dose NTG was not changed, a strong reduction in vasodilator-stimulated phosphoprotein (VASP) serine239 phosphorylation (specific substrate of cGK-I) was observed in tolerant tissue from rats and rabbits. Concomitant in vivo and in vitro treatment with vitamin C improved tolerance, reduced oxidative stress, and improved P-VASP. Conclusions —We therefore conclude that increased expression of sGC in the setting of tolerance reflects a chronic inhibition rather than an induction of the sGC–cGK-I pathway and may be mediated at least in part by increased vascular superoxide.

Background Risk factors for atherosclerosis such as hypercholesterolemia and hypertension are associated with endothelial dysfunction of conduit and resistance vessels; however, the interaction of these risk factors and underlying mechanisms affecting endothelial function remain to be determined. The present study investigated the role of long-term smoking and hypercholesterolemia and their impact on endothelial function of peripheral resistance vessels in relation to plasma levels of autoantibodies against oxidized LDL, which has been implicated in the development of endothelial dysfunction and atherosclerosis. Methods and Results The vascular responses to the endothelium-dependent agent acetylcholine (7.5, 15, 30, and 60 μg/min) and the endothelium-independent agent sodium nitroprusside (1, 3, and 10 μg/min) were studied in normal control subjects (n=10), patients with hypercholesterolemia (n=15), long-term smokers (n=15), and hypercholesterolemic patients who smoked (n=15). Drugs were infused into the brachial artery, and forearm blood flow (FBF) was measured by venous occlusion plethysmography. The FBF responses to acetylcholine were significantly blunted in all three patient groups compared with normal control subjects ( P P r =−.53, P Conclusions These results demonstrate that cigarette smoking and hypercholesterolemia synergistically impair endothelial function and that their combined presence is associated with increased plasma levels of autoantibodies against oxidized LDL. These observations raise the possibility that long-term smoking potentiates endothelial dysfunction in hypercholesterolemic patients by enhancing the oxidation of LDL.

BACKGROUND The purpose of this study was to investigate the therapeutic potential of prolonged inhibition of atrial natriuretic factor (ANF) degradation in patients with severe chronic heart failure. METHODS AND RESULTS The effects of repeated doses of the endopeptidase inhibitor candoxatrilat (150 mg i.v.) were examined over a 24-hour period in patients with severe chronic heart failure (New York Heart Association class III-IV). Plasma alpha-hANF(99-126) was elevated at baseline (235 +/- 59 pg/ml), increased 2.5-fold at 2 hours after the first dose, and remained significantly elevated throughout the 24-hour protocol. In contrast, pro-hANF(31-67) decreased from 3,151 +/- 616 to 2,072 +/- 362 pg/ml (p less than 0.05). Cardiac index (CI) increased only transiently after the first dose of candoxatrilat (CI, 2.11 +/- 0.2 to 2.67 +/- 0.28 l/min/m2, p less than 0.05). Sodium excretion increased sixfold (p less than 0.05) 2 hours after the first dose of candoxatrilat and remained significantly elevated throughout the protocol. Degree of natriuresis and diuresis in response to candoxatrilat was closely related to baseline cardiac output. Glomerular filtration rate and volume excretion did not change significantly. Pulmonary capillary wedge pressure fell from 23 +/- 3 to 18 +/- 3 mm Hg (p less than 0.05) and remained below baseline throughout the 24 hours. Arterial pressure, heart rate, and total peripheral resistance did not change significantly during the 24-hour period. Urinary cGMP excretion increased fivefold (p less than 0.05), whereas urinary ANF immunoreactivity and plasma cGMP levels remained unchanged. Excretion of prostacyclin metabolite 6-keto-PGF-1 alpha increased 3.3-fold (p less than 0.05). Plasma norepinephrine and epinephrine levels decreased significantly after candoxatrilat and remained suppressed over the 24-hour period. There was also a transient reduction in plasma vasopressin, aldosterone levels, and plasma renin activity. Hematocrit, total protein content, and plasma albumin concentrations did not change, indicating that no fluid shift into the extravascular space had occurred. CONCLUSIONS 1) The inhibition of ANF degradation causes sustained drop in left and right atrial pressures that appears to be mediated by an inhibition of neurohumoral activity; 2) concomitant inhibition of bradykinin breakdown (which in turn stimulates renal prostacyclin synthesis) contributes to natriuresis; 3) the close correlation between renal response and baseline cardiac index indicates that an inadequate renal perfusion secondary to low cardiac output diminishes the efficacy of this treatment modality. This spectrum of action would be advantageous for a first-line diuretic agent early in the course of disease rather than in patients with advanced chronic heart failure.

The Holt-Oram syndrome is an autosomal dominant heritable disorder characterized by skeletal upper-limb dysplasias and congenital cardiac defects. We describe a 43-year-old woman who presented with paroxysmal tachycardia and progressive heart failure. Both ring fingers were abnormally short as a result of dysplasia of metacarpal IV (Figure 1⇓). Auscultation revealed a loud systolic murmur at the left sternal margin and a widely split S2. The ECG was consistent with right ventricular hypertrophy (Figure 2⇓). The chest …

The feedback control of neuroendocrine activity by cardiopulmonary blood volume is disturbed in congestive heart failure. By analyzing plasma catecholamine kinetics, we tested in 11 chronically instrumented conscious dogs whether attenuations in the sympathoadrenal inhibition induced by atrial natriuretic peptide (ANP) contributed to this disturbance. Low-output failure was brought about by continuous ventricular pacing at 265 beats/min for 2 weeks. This resulted in a decline in aortic flow by 37 +/- 5% (SEM), an increase in peripheral vascular resistance by 48 +/- 4%, a 13 +/- 3-fold elevation in plasma ANP, a 9 +/- 3-fold elevation in plasma renin activity, and an augmentation of the norepinephrine-release rate into plasma by 132 +/- 17%. During ANP infusion, the epinephrine-release rate declined by 26 +/- 5% per 10-fold elevation in plasma ANP before pacing and by 31 +/- 7% (not significantly different) after 2 weeks of pacing. Before pacing, ANP attenuated plasma renin activity and caused hypotension without a rise in norepinephrine-release rate. After 2 weeks of pacing, ANP lowered norepinephrine release (by 16 +/- 6%) without affecting blood pressure or plasma renin activity, and vascular nonresponsiveness to ANP was verified under autonomic blockade. These data indicate that, during the development of heart failure, an inhibitory action of ANP on norepinephrine release is unmasked by an ANP-specific vascular desensitization, whereas the inhibition of epinephrine release is observed throughout. It is concluded that ANP-induced sympathoadrenal inhibition is not attenuated and, therefore, does not contribute to the disturbed regulation observed early in the development of failure.

N-acetylcysteine is assumed to reverse nitrate tolerance by replenishing depleted intracellular sulfhydryl groups, but data on interactions of N-acetylcysteine and nitrates in patients with stable angina are controversial and disappointing. Therefore, we studied the effect of N-acetylcysteine on nitrate responsiveness of epicardial arteries and of the venous system (assessed as changes in effective vascular compliance) in dogs (n = 12) during long-term nitroglycerin treatment (1.5 micrograms/kg/min i.v. for 5-6 days). In dogs with nitroglycerin-specific tolerance (shift of venous or epicardial artery dilation to 15-17-fold higher dosages), N-acetylcysteine (100 mg/kg i.v.) had no dilator effect and did not alter the dose-response relations of nitroglycerin. Yet, in nontolerant dogs (n = 17), N-acetylcysteine augmented (1.5-2.0-fold) the dilation of epicardial arteries and the reduction of peripheral vascular resistance induced by 0.5-1.5 micrograms/kg/min nitroglycerin. In vitro, the augmentation of purified guanylate cyclase activity by nitroglycerin (10-100 microM) was potentiated by N-acetylcysteine (0.01-1.0 mM) in saline or in canine plasma, but N-acetylcysteine alone was ineffective. We conclude that 1) N-acetylcysteine does not restore nitroglycerin responsiveness in tolerant epicardial arteries or veins in vivo, 2) a small, tolerance-independent augmentation of nitroglycerin-induced dilation may result from N-acetylcysteine-induced extracellular formation of a stimulant of guanylate cyclase from nitroglycerin.