Petra Kleinbongard

Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals

Changes in plasma nitrite concentration in the human forearm circulation have recently been shown to reflect acute changes in endothelial nitric oxide synthase (eNOS)-activity. Whether basal plasma nitrite is a general marker of constitutive NOS-activity in vivo is yet unclear. Due to the rapid metabolism of nitrite in blood and the difficulties in its analytical determination literature data on levels of nitrite in mammals are largely inconsistent. We hypothesized that constitutive NOS-activity in the circulatory system is relatively uniform throughout the mammalian kingdom. If true, this should result in comparable systemic plasma nitrite levels in different species. Using three different analytical approaches we determined plasma nitrite concentration to be in a nanomolar range in a variety of species: humans (305 +/- 23 nmol/l), monkeys (367 +/- 62 nmol/l), minipigs (319 +/- 24 nmol/l), dogs (305 +/- 50 nmol/l), rabbits (502 +/- 21 nmol/l), guinea pigs (412 +/- 44 nmol/l), rats (191 +/- 43 nmol/l), and mice (457 +/- 51 nmol/l). Application of different NOS-inhibitors in humans, minipigs, and dogs decreased NOS-activity and thereby increased vascular resistance. This was accompanied by a significant, up to 80%, decrease in plasma nitrite concentration. A comparison of plasma nitrite concentrations between eNOS(-/-) and NOS-inhibited wild-type mice revealed that 70 +/- 5% of plasma nitrite is derived from eNOS. These results provide evidence for a uniform constitutive vascular NOS-activity across mammalian species.

Coronary Microembolization From Bedside to Bench and Back to Bedside

Coronary microembolization from the erosion or rupture of a vulnerable atherosclerotic plaque occurs spontaneously in acute coronary syndromes and iatrogenically during percutaneous coronary interventions. Typical consequences of coronary microembolization are microinfarcts with an inflammatory response, contractile dysfunction, and reduced coronary reserve. Apart from transient elevations of creatine kinase and troponin, microemboli can be visualized by intracoronary Doppler and the resulting microinfarcts by late-enhancement nuclear magnetic resonance. Statins, antiplatelet agents, and coronary vasodilators protect against microembolization and microinfarction when started before percutaneous coronary interventions. Distal protection devices can retrieve atherothrombotic debris and prevent its embolization into the microcirculation, but their effect on clinical outcome has been disappointing so far, except for saphenous vein bypass grafts. Devices for aspiration of thrombi and thrombus-derived vasoconstrictor, thrombogenic, and inflammatory substances, however, reduce thrombus burden, improve perfusion, and provide protection in patients with acute myocardial infarction.

Cardioprotective and prognostic effects of remote ischaemic preconditioning in patients undergoing coronary artery bypass surgery: a single-centre randomised, double-blind, controlled trial

BACKGROUND Remote ischaemic preconditioning has been associated with reduced risk of myocardial injury after coronary artery bypass graft (CABG) surgery. We investigated the safety and efficacy of this procedure. METHODS Eligible patients were those scheduled to undergo elective isolated first-time CABG surgery under cold crystalloid cardioplegia and cardiopulmonary bypass at the West-German Heart Centre, Essen, Germany, between April, 2008, and October, 2012. Patients were prospectively randomised to receive remote ischaemic preconditioning (three cycles of 5 min ischaemia and 5 min reperfusion in the left upper arm after induction of anaesthesia) or no ischaemic preconditioning (control). The primary endpoint was myocardial injury, as reflected by the geometric mean area under the curve (AUC) for perioperative concentrations of cardiac troponin I (cTnI) in serum in the first 72 h after CABG. Mortality was the main safety endpoint. Analysis was done in intention-to-treat and per-protocol populations. This trial is registered with ClinicalTrials.gov, number NCT01406678. FINDINGS 329 patients were enrolled. Baseline characteristics and perioperative data did not differ between groups. cTnI AUC was 266 ng/mL over 72 h (95% CI 237-298) in the remote ischaemic preconditioning group and 321 ng/mL (287-360) in the control group. In the intention-to-treat population, the ratio of remote ischaemic preconditioning to control for cTnI AUC was 0·83 (95% CI 0·70-0·97, p=0·022). cTnI release remained lower in the per-protocol analysis (0·79, 0·66-0·94, p=0·001). All-cause mortality was assessed over 1·54 (SD 1·22) years and was lower with remote ischaemic preconditioning than without (ratio 0·27, 95% CI 0·08-0·98, p=0·046). INTERPRETATION Remote ischaemic preconditioning provided perioperative myocardial protection and improved the prognosis of patients undergoing elective CABG surgery. FUNDING German Research Foundation.

TNFα in atherosclerosis, myocardial ischemia/reperfusion and heart failure

TNFalpha is crucially involved in the pathogenesis and progression of atherosclerosis, myocardial ischemia/reperfusion injury and heart failure. The formation and release of TNFalpha and its downstream signal transduction cascade following activation of its two receptor subtypes is characterized, with special emphasis on the cardiovascular system. In the vasculature, TNFalpha alters endothelial and vascular smooth muscle cell function as well as endothelial cell-blood cell interaction; the importance of such alterations for vascular dysfunction, the initiation and progression of atherosclerosis are discussed. In the myocardium, TNFalpha contributes to reversible and irreversible ischemia/reperfusion injury, post-myocardial infarction remodeling and heart failure development. Simultaneously, TNFalpha also contributes to cardioprotection by ischemic conditioning. Emphasis is placed on such ambivalent (detrimental vs. beneficial) role of TNFalpha, which appears to be dose- and time-dependent and in part related to the activation of the specific receptor subtype. Given the ambivalent role of TNFalpha and its receptors, it is not surprising that clinical trials using compounds that antagonize TNFalpha revealed ambiguous and largely disappointing results in cardiovascular disease, notably in heart failure. Future perspectives to antagonize and/or potentially recruit TNFalpha in the cardiovascular system are critically discussed.

Evidence for in vivo transport of bioactive nitric oxide in human plasma

Although hitherto considered as a strictly locally acting vasodilator, results from recent clinical studies with inhaled nitric oxide (NO) indicate that NO can exert effects beyond the pulmonary circulation. We therefore sought to investigate potential remote vascular effects of intra-arterially applied aqueous NO solution and to identify the mechanisms involved. On bolus application of NO into the brachial artery of 32 healthy volunteers, both diameter of the downstream radial artery and forearm blood flow increased in a dose-dependent manner. Maximum dilator responses were comparable to those after stimulation of endogenous NO formation with acetylcholine and bradykinin. Response kinetics and pattern of NO decomposition suggested that despite the presence of hemoglobin-containing erythrocytes, a significant portion of NO was transported in its unbound form. Infusion of NO (36 micromol/min) into the brachial artery increased levels of plasma nitroso species, nitrite, and nitrate in the draining antecubital vein (by < 2-fold, 30-fold, and 4-fold, respectively), indicative of oxidative and nitrosative chemistry. Infused N-oxides were inactive as vasodilators whereas S-nitrosoglutathione dilated conduit and resistance arteries. Our results suggest that NO can be transported in bioactive form for significant distances along the vascular bed. Both free NO and plasma nitroso species contribute to the dilation of the downstream vasculature.

Sustained increase in flow-mediated dilation after daily intake of high-flavanol cocoa drink over 1 week.

A single-dose ingestion of flavanol-rich cocoa acutely reverses endothelial dysfunction. To investigate the time course of endothelial function during daily consumption of high-flavanol cocoa, we determined flow-mediated dilation (FMD) acutely (for up to 6 hours after single-dose ingestion) and chronically (administration for 7 days). The study population represented individuals with smoking-related endothelial dysfunction; in addition to FMD, plasma nitrite and nitrate were measured. The daily consumption of a flavanol-rich cocoa drink (3 × 306 mg flavanols/d) over 7 days (n = 6) resulted in continual FMD increases at baseline (after overnight fast and before flavanol ingestion) and in sustained FMD augmentation at 2 hours after ingestion. Fasted FMD responses increased from 3.7 ± 0.4% on day 1 to 5.2 ± 0.6%, 6.1 ± 0.6%, and 6.6 ± 0.5% (each P < 0.05) on days 3, 5, and 8, respectively. FMD returned to 3.3 ± 0.3% after a washout week of cocoa-free diet (day 15). Increases observed in circulating nitrite, but not in circulating nitrate, paralleled the observed FMD augmentations. The acute, single-dose consumption of cocoa drinks with 28 to 918 mg of flavanols led to dose-dependent increases in FMD and nitrite, with a maximal FMD at 2 hours after consumption. The dose to achieve a half-maximal FMD response was 616 mg (n = 6). Generally applied biomarkers for oxidative stress (plasma, MDA, TEAC) and antioxidant status (plasma ascorbate, urate) remained unaffected by cocoa flavanol ingestion. The daily consumption of flavanol-rich cocoa has the potential to reverse endothelial dysfunction in a sustained and dose-dependent manner.

Plasma Nitrosothiols Contribute to the Systemic Vasodilator Effects of Intravenously Applied NO Experimental and Clinical Study on the Fate of NO in Human Blood

Abstract— Higher doses of inhaled NO exert effects beyond the pulmonary circulation. How such extrapulmonary effects can be reconciled with the presumed short half-life of NO in the blood is unclear. Whereas erythrocytes have been suggested to participate in NO transport, the exact role of plasma in NO delivery in humans is not clear. Therefore, we investigated potential routes of NO decomposition and transport in human plasma. NO consumption in plasma was accompanied by a concentration-dependent increase in nitrite and S-nitrosothiols (RSNOs), with no apparent saturation limit up to 200 &mgr;mol/L. The presence of red blood cells reduced the formation of plasma RSNOs. Intravenous infusion of 30 &mgr;mol/min NO in healthy volunteers increased plasma levels of RSNOs and induced systemic hemodynamic effects at the level of both conduit and resistance vessels, as reflected by dilator responses in the brachial artery and forearm microvasculature. Intravenous application of S-nitrosoglutathione, a potential carrier of bioactive NO, mimicked the vascular effects of NO, whereas nitrite and nitrate were inactive. Changes in plasma nitrosothiols were correlated with vasodilator effects after intravenous application of S-nitrosoglutathione and NO. These findings demonstrate that in humans the pharmacological delivery of NO solutions results in the transport and delivery of NO as RSNOs along the vascular tree.

TNFα in myocardial ischemia/reperfusion, remodeling and heart failure

TNFα is crucially involved in the pathogenesis and progression of myocardial ischemia/reperfusion injury and heart failure. The formation and release of TNFα and its downstream signal transduction cascade following activation of its two receptor subtypes are characterized. Myocardial TNFα and TNF receptor activation have an ambivalent role in myocardial ischemia/reperfusion injury and protection from it. Excessive TNFα expression and subsequent cardiomyocyte TNF receptor type 1 stimulation induce contractile dysfunction, hypertrophy, fibrosis and cell death, while a lower TNFα concentration and subsequent cardiomyocyte TNF receptor type 2 stimulation are protective. Apart from its concentration and receptor subtype, the myocardial action of TNFα depends on the duration of its exposure and its localization. While detrimental during sustained ischemia, TNFα contributes to ischemic preconditioning protection, no matter whether it is the first, second or third window of protection, and both TNF receptors are involved in the protective signal transduction cascade. Finally, the available clinical attempts to antagonize TNFα in cardiovascular disease, notably heart failure, are critically discussed.

Across-Species Transfer of Protection by Remote Ischemic Preconditioning With Species-Specific Myocardial Signal Transduction by Reperfusion Injury Salvage Kinase and Survival Activating Factor Enhancement Pathways

RATIONALE Reduction of myocardial infarct size by remote ischemic preconditioning (RIPC), that is, cycles of ischemia/reperfusion in an organ remote from the heart before sustained myocardial ischemia/reperfusion, was confirmed in all species so far, including humans. OBJECTIVE To identify myocardial signal transduction of cardioprotection by RIPC. METHODS AND RESULTS Anesthetized pigs were subjected to RIPC (4×5/5 minutes hindlimb ischemia/reperfusion) or placebo (PLA) before 60/180 minutes coronary occlusion/reperfusion. Phosphorylation of protein kinase B, extracellular signal-regulated kinase 1/2 (reperfusion injury salvage kinase [RISK] pathway), and signal transducer and activator of transcription 3 (survival activating factor enhancement [SAFE] pathway) in the area at risk was determined by Western blot. Wortmannin/U0126 or AG490 was used for pharmacological RISK or SAFE blockade, respectively. Plasma sampled after RIPC or PLA, respectively, was transferred to isolated bioassay rat hearts subjected to 30/120 minutes global ischemia/reperfusion. RIPC reduced infarct size in pigs to 16±11% versus 43±11% in PLA (% area at risk; mean±SD; P<0.05). RIPC increased the phosphorylation of signal transducer and activator of transcription 3 at early reperfusion, and AG490 abolished the protection, whereas RISK blockade did not. Signal transducer and activator of transcription 5 phosphorylation was decreased at early reperfusion in both RIPC and PLA. In isolated rat hearts, pig plasma taken after RIPC reduced infarct size (25±5% of ventricular mass versus 38±5% in PLA; P<0.05) and activated both RISK and SAFE. RISK or SAFE blockade abrogated this protection. CONCLUSIONS Cardioprotection by RIPC in pigs causally involves activation of signal transducer and activator of transcription 3 but not of RISK. Protection can be transferred with plasma from pigs to isolated rat hearts where activation of both RISK and SAFE is causally involved. The myocardial signal transduction of RIPC is the same as that of ischemic postconditioning.

Nitric oxide differentially regulates proliferation and mobilization of endothelial progenitor cells but not of hematopoietic stem cells

To investigate the role of nitric oxide in controlling endothelial progenitor (EPC) and hematopoietic stem cell (HSC) mobilization, wild-type mice, L-NAME treated WT and eNOS-/- mice received either PBS or G-CSF for 5 days. Under unstimulated conditions bone marrow of either L-NAME treated WT and eNOS-/- mice, representing acute and chronic NO-deficiency, showed higher CD34(+)Flk-I+ EPC numbers compared to their WT littermates. Furthermore, CD34(+)Flk-I+ progenitors under NO-deficient conditions showed a higher cell turn over since the proliferation and apoptosis activity under in vivo as well as in vitro conditions were enhanced. In line with this finding bone marrow derived EPC differentiation towards endothelial cells was modulated in an NO-dependent manner. Administration of G-CSF resulted in an increase of EPC within the bone marrow of WT animals with a consecutive release of these cells into the peripheral circulation. Under NO-deficient conditions G-CSF failed to increase EPC numbers. In contrast, the HSC population c-kit(+)Lin- was not influenced by nitric oxide. Thus, NO differentially supports the mobilization of the endothelial committed progenitor subpopulation in bone marrow but does not have an effect on HSC in vivo.