Georg Kojda

Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure

There is a growing body of evidence suggesting that numerous pathological conditions are associated with increased vascular production of reactive oxygen species. This form of vascular oxidant stress and particularly interactions between NO and oxygen-derived radicals represent a common pathological mechanism present in many so-called risk factors for atherosclerosis. Furthermore, reactive oxygen species seem to serve important cellular signalling mechanisms responsible for many of the features of vascular lesion formation. The mechanisms whereby vascular cells produce reactive oxygen species are only presently coming to light, and almost certainly will prove to be a focus for future therapies.

Vasoprotection by nitric oxide: mechanisms and therapeutic potential

Endothelial production of nitric oxide (nitrogen monoxide, NO) has become a major research area in vascular biology. Some of the most important effects that NO exerts in the vascular wall are potentially vasoprotective, because these effects maintain important physiological functions such as vasodilation, anticoagulation, leucocyte adhesion, smooth muscle proliferation, and the antioxidative capacity. During the last 2 decades it has become apparent that a variety of diseases are associated with an impairment of endothelium-dependent NO activity. One of the major causes is believed to be an increased production of reactive oxygen species, in particular superoxide, which have been shown to interfere with many steps of the NO--cyclic guanosine monophosphate (cGMP) pathway. This phenomenon has been found in diverse conditions such as atherosclerosis, hypertension, diabetes, hypercholesterolemia, heart failure, and cigarette smoking. The aim of this review is to examine the cellular and molecular mechanisms whereby NO exerts potentially vasoprotective effects and to discuss pharmacologic approaches targeting the NO pathway in view of their potential to improve endothelial function and to reduce the progression of atherosclerotic vascular disease. We conclude that there is compelling evidence for vasoprotective actions of NO which are mediated by cGMP-dependent and cGMP-independent mechanisms. These effects may contribute to the beneficial effects of established drugs such as ACE inhibitors or statins. Unfortunately, clinical data on the effect of long-term treatment with nitrates on the progression of coronary artery disease are lacking. Finally, L-arginine or new activators of the NO pathway may become therapeutic options in the future.

Low Increase in cGMP Induced by Organic Nitrates and Nitrovasodilators Improves Contractile Response of Rat Ventricular Myocytes

Whether organic nitrates are bioactivated to NO in cardiac muscle cells and may thus directly affect cardiac contractile function has remained an open question. Therefore, we determined the effects of the organic nitrates glyceryl trinitrate (100 mumol/L), pentaerythritol tetranitrate (10 mumol/L), and isosorbide-5-mononitrate on electrically stimulated contractile response (CR) and cAMP and cGMP content of isolated adult rat ventricular cardiomyocytes compared with different concentrations of the spontaneous NO donors S-nitroso-N-acetyl-d,1-penicillamine (SNAP) and 2,2-diethyl-1-hydroxy-1-nitroso-hydrazine (DEA/NO). A high concentration of spontaneous NO donors (100 mumol/L caused a large increase in cGMP content that was accompanied by a decrease in CR to 73.8 +/- 6.7% (SNAP) and 80.9 +/- 6.1% (DEA/NO) of the control values. Inhibition of cGMP-dependent protein kinase by 10 mumol/L KT 5822 converted this effect into a pronounced improvement of CR (163.5 +/- 14.0%) By contrast, the organic nitrates caused a small but significant increase in cGMP, which was accompanied by an increase in cAMP and CR identical to that induced by 10 nmol/L isoprenaline (141.6 +/- 6.4%) A similar effect was observed with a low concentration (1 mumol/L of SNAP and DEA/NO. All increases in CR induce by nitrates were abolished after inhibition of cAMP-dependent protein kinase by Rp-cAMPS (10 mumol/L). The positive contractile effect of isoprenaline was enhanced by 1 mumol/L SNAP. This effect was also demonstrated in isolated rat papillary muscles. These results indicate that in cardiac muscle (1) organic nitrate are bioactivated to NO; (2) this results in a moderate increase in cGMP, which causes an improved CR by increasing cAMP and activating cAMP-dependent protein kinase; and (3) a large increase in cGMP, produced by high doses of NO donors, reduces CR because of the activation of CGMP-dependent protein kinase.

Regulation of basal myocardial function by NO

The effects of exogenous and endogenous. NO on myocardial functions such as contraction, relaxation and heart rate have recently gained considerable scientific interest. .NO stimulates myocardial soluble guanylate cyclase to produce cGMP, which activates two major target proteins. A small increase in cGMP levels predominantly inhibits phosphodiesterase III, while high cGMP levels activate cGMP-dependent protein kinase. Accordingly, submicromolar .NO concentrations improve myocardial contraction, while submillimolar .NO concentrations decrease contractility. The latter action includes direct inhibitory .NO effects on ATP synthesis and voltage-gated calcium channels. Overall, the inotropic effects of exogenous .NO are small and probably of minor importance for myocardial contractility. Cardiomyocytes are capable of expressing eNOS and iNOS. Endogenous .NO has effects on myocardial contraction, similar to that of exogenous .NO. Various NOS inhibitors can substantially reduce myocardial contractility in vitro and in vivo, suggesting that basal endogenous .NO production supports myocardial contractility. There is also evidence for a .NO-dependent cardiodepressive effect of cytokines that is mediated by expression of iNOS. This is consistent with the negative inotropic effects of .NO at high concentrations. Cardiodepressive actions of endogenous .NO production may play a role in certain forms of heart failure. Finally, .NO also has an effect on heart rate. Physiologic .NO concentrations can stimulate heart rate by activating the hyperpolarization-activated inward current (If) and this effect decreases at submillimolar .NO concentrations. In summary, physiological concentrations of .NO increase contractility and heart rate under basal conditions, while high .NO concentrations induce the opposite effects.

Nonallergic angioedema: role of bradykinin

Angioedema is an underestimated clinical problem. Many cases are nonallergic reactions, e.g. bradykinin‐induced angioedema caused by genetic defects and angiotensin‐converting enzyme (ACE) inhibitors. This difference is crucial for successful therapy, in particular when complete emergency care is not available. Five important forms of nonallergic angioedema can be distinguished: hereditary (HAE), acquired (AAE), renin‐angiotensin‐aldosterone system (RAAS)‐blocker‐induced (RAE), pseudoallergic angioedema (PAE) and idiopathic angioedema (IAE). Some angioedema are present in the larynx and may cause death. A vast majority of nonallergic angioedema are RAE, particularly those caused by ACE inhibitors. It appears important to emphasize that in patients with complete intolerance to RAAS‐blockers, cessation of RAAS‐blockers is likely to be associated with increased cardiovascular risk. Currently, there is no published algorithm for diagnosis and treatment. Angioedema is usually treated by a conservative clinical approach using artificial ventilation, glucocorticoids and antihistamines. Today, a plasma pool C1‐esterase inhibitor (C1‐INH) concentrate is the therapy of choice in HAE. The current pharmacotherapy of nonallergic angioedema is not satisfactory, thus requiring the identification of effective agents in clinical trials. Recently, several new drugs were developed: a recombinant C1‐INH, a kallikrein inhibitor (ecallantide) and a specific bradykinin‐B2‐receptor antagonist (icatibant). According to currently available reports, these drugs may improve the treatment of kinin‐induced angioedema.

Therapeutic Efficacy of Icatibant in Angioedema Induced by Angiotensin-Converting Enzyme Inhibitors: A Case Series

STUDY OBJECTIVE The pathophysiology of angiotensin-converting enzyme inhibitor (ACEi)-induced angioedema most likely resembles that of hereditary angioedema, ie, it is mainly mediated by bradykinin-induced activation of vascular bradykinin B2 receptors. We hypothesize that the bradykinin B2 receptor antagonist icatibant might be an effective therapy for ACEi-induced angioedema. METHODS Eight patients with acute ACEi-induced angioedema were treated with a single subcutaneous injection of icatibant. The outcome was assessed by the time to first improvement of symptoms, complete symptom relief, and drug safety. In addition, we retrospectively assessed the clinical course of 47 consecutive patients of our clinic with ACEi-induced angioedema. RESULTS First symptom improvement after icatibant injection occurred at a mean time of 50.6 minutes (standard deviation [SD] 21 minutes) and complete relief of symptoms at 4.4 hours (SD 0.8 hours). No patient received tracheal intubation, other drug treatment, tracheotomy, or a second icatibant injection. There were no adverse effects except erythema occurring at the injection site. In the historical comparison group treated with methylprednisolone and clemastine, the mean time to complete relief of symptoms was 33 hours (SD 19.4 hours). Some of these patients received a tracheotomy (3/47), were intubated (2/47), or received a second dose of methylprednisolone (12/47). CONCLUSION Although sample size limits the external validity of our results, the substantial decrease of time to complete symptom relief suggests that this new treatment is likely effective as a pharmacotherapeutic approach to treat ACEi-induced angioedema.

New topics in bradykinin research

To cite this article: Maurer M, Bader M, Bas M, Bossi F, Cicardi M, Cugno M, Howarth P, Kaplan A, Kojda G, Leeb‐Lundberg F, Lötvall J, Magerl M. New topics in bradykinin research. Allergy 2011; 66: 1397–1406.

The effect of peroxynitrite on the catalytic activity of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a key enzyme of the *NO/cGMP pathway. Many cardiovascular disorders are associated with reduced *NO-mediated effects, while vascular superoxide (O(2)*(-)) production is increased. Both radicals rapidly react to peroxynitrite. We investigated whether peroxynitrite affects the activity and protein expression of sGC in intact vascular preparations. Catalytic sGC activity and expression of the sGC-beta(1) subunit was measured by conversion of radiolabeled GTP and western blot, respectively, using cytosolic extracts from rat aorta that had been incubated for 4 h with *NO/O(2)*(-) systems (devoid of free *NO) generating either 0.13 microM or 7.5 microM peroxynitrite/min. Incubation of rat aorta with 0.13 microM peroxynitrite/min had no effect. In striking contrast, incubation with 7.5 microM peroxynitrite/min resulted in a shift of the concentration-response curve obtained with a *NO donor (p =.0004) and a reduction of maximal specific activity from 3579 +/- 495 to 2422 +/- 265 pmol cGMP/mg/min (p =.036). The expression of the sGC-beta(1) subunit was unchanged. Exposure of aorta to the O(2)*(-) component had no effect, while exposure to the *NO-component reduced sGC expression to 58.8 +/- 7% (p <.001) and maximal sGC activity from 4041 +/- 992 to 1429 +/- 491 pmol cGMP/mg/min (p =.031). These data suggest that continuous generation of extracellular peroxynitrite might interfere with the *NO/cGMP signaling in vascular cells.

Alzheimer’s disease, cerebrovascular dysfunction and the benefits of exercise: From vessels to neurons

Exercise training promotes extensive cardiovascular changes and adaptive mechanisms in both the peripheral and cerebral vasculature, such as improved organ blood flow, induction of antioxidant pathways, and enhanced angiogenesis and vascular regeneration. Clinical studies have demonstrated a reduction of morbidity and mortality from cardiovascular disease among exercising individuals. However, evidence from recent large clinical trials also suggests a substantial reduction of dementia risk - particularly regarding Alzheimers disease (AD) - with regular exercise. Enhanced neurogenesis and improved synaptic plasticity have been implicated in this beneficial effect. However, recent research has revealed that vascular and specifically endothelial dysfunction is essentially involved in the disease process and profoundly aggravates underlying neurodegeneration. Moreover, vascular risk factors (VRFs) are probably determinants of incidence and course of AD. In this review, we emphasize the interconnection between AD and VRFs and the impact of cerebrovascular and endothelial dysfunction on AD pathophysiology. Furthermore, we describe the molecular mechanisms of the beneficial effects of exercise on the vasculature such as activation of the vascular nitric oxide (NO)/endothelial NO synthase (eNOS) pathway, upregulation of antioxidant enzymes, and angiogenesis. Finally, recent prospective clinical studies dealing with the effect of exercise on the risk of incident AD are briefly reviewed. We conclude that, next to upholding neuronal plasticity, regular exercise may counteract AD pathophysiology by building a vascular reserve.

In vivo effects of pentaerythrityl-tetranitrate and isosorbide-5-mononitrate on the development of atherosclerosis and endothelial dysfunction in cholesterol-fed rabbits.

We wished to determine whether long-term treatment with organic nitrovasodilators has pharmacological effects on the development of atherosclerotic lesions and endothelial dysfunction in cholesterol-fed rabbits. For 15 weeks, six groups of 9 New Zealand White rabbits received a standard diet, which contained no admixture, pentaerythrityl-tetranitrate (PETN 6 mg/kg body weight/day), or isosorbide-5-mononitrate (ISMN 2 mg/kg body weight/day). In the other three groups, the same diets were further enriched with cholesterol (0,75%). Four rings of thoracic aorta were used for tension studies; these rings and the aortas from the aortic arch to bifurcation were then fixed in formol and stained with Sudan IV to determine the area of luminal atherosclerotic lesions by a computerized laser-scanning approach. The cholesterol diet increased plasma levels of cholesterol from 69.8 ± 10.4 to 907.1 ± 85.5 mg/dl. A similar result was obtained in the group receiving PETN/cholesterol, but the group fed ISMN/cholesterol showed a significantly higher plasma level of cholesterol (1.165 ± 81.4 mg/dl). Plasma levels of PETN metabolites were still detectable by gas chromatography/mass spectrometry after a 24-h in vivo washout period. The cholesterol diet induced a pronounced degree of atherosclerotic lesions in the aortic arch and the thoracic and abdominal aorta: 73.3 ± 1.9. 46.3 ± 2.5, and 49.6 ± 3.6%, respectively. Additional treatment with PETN resulted in a reduction of this atherosclerotic area to 58.6 ± 2.05% (p < 0.0001), 34.7 ± 1.98% (p < 0.01), and 39.3 ± 3.06% (p < 0.05). In contrast, ISMN had no significant effect on this parameter. The cholesterol diet also induced an endothelial dysfunction as indicated by the diminished vasorelaxation induced by acetylcholine (ACh). Treatment with PETN completely inhibited the development of endothelial dysfunction, whereas ISMN had no effect. In the three groups receiving a cholesterol diet, an increased extent of aortic lesions significantly correlated with increased endothelial dysfunction measured in the same preparations. The long-term treatment with PETN did not affect the vasorelaxing potency of PETN in aortic rings, and similar results were obtained in the case of ISMN. We conclude that long-term treatment with doses of PETN, which do not promote the development of in vitro vascular nitrate tolerance, may protect against atherosclerosis and endothelial dysfunction. This novel, yet unknown pharmacodynamic quality of nitrovasodilators like PETN may contribute to their long-term efficacy in coronary artery disease but may also imply new therapeutic indications in the future.