Detektion von reaktiven Sauerstoffspezies an isoliert perfundiert und ventilierten Kaninchenlungen mittels Elektronenspinresonanz-Spektroskopie

Reaktive Sauerstoffspezies (ROS) spielen eine wichtige Rolle in biologischen Systemen. ROS gewinnen bei einer Reihe von Krankheiten, aber auch bei physiologischen Regulationsmechanismen, immer mehr an Bedeutung. Deshalb ist es wichtig, ihre Rolle in pathophysiologischen Prozessen, wie z.B. beim ARDS (Adult Respiratory Distress Syndrome), bei Sepsis, beim Ischämie/Reperfusionsschaden und bei Signaltransduktionsmechanismen in biologischen Systemen aufzuklären. Im pulmonalen System wird vermutet, dass reaktive Sauerstoffspezies unter Hypoxie am Mechanismus der Ventilations-Perfusions-Anpassung beteiligt sind. Jedoch ist unklar, ob ROS während alveolärer Hypoxie vermindert oder paradoxerweise vermehrt freigesetzt werden und von welcher Quelle die ROS gebildet werden. Als Quellen kommen u.a. Mitochondrien und NADPH-Oxidasen in Betracht. Im Rahmen dieser Arbeit wurde eine Methode zur Detektion von intravaskulär freigesetzten ROS an der isoliert ventilierten und perfundierten Kaninchenlungen etabliert. Hierbei stellte die Kombination aus Elektronspinresonazspektroskopie (ESR-Spektroskopie), Spin-Trapping-Technik und der Methode der isolierten Lunge eine neue Entwicklung dar. Als „Spinprobe“ wurde in der vorliegenden Arbeit das zyklische Hydroxylamin l-Hydroxy-3-Carboxy-2,2,5,5-Tetramethylpyrrolidin (CPH) benutzt und charakterisiert. CPH wurde in den letzten Jahren zur quantitativen Messung von reaktiven Sauerstoffspezies in biologischen Systemen eingeführt. ROS können mit der Detektion des korrespondierenden Radikals 3-Carboxy-Proxyl (CP.) mittels ESR quantifiziert werden. Durch ESR-Spektroskopie konnte das triple-line Spektrum des CP. Radikals detektiert werden. CPH hat die Eigenschaft, dass es auch ohne Anwesenheit von Radikalen durch Sauerstoff oxidiert werden kann. Dieser Vorgang wird als Autooxidation bezeichnet. Aus diesem Grund wurde zunächst eine Charakterisierung der Autooxidation notwendig. Um eine mögliche Sauerstoffabhängigkeit der Superoxidfreisetzung zu untersuchen, wurden die isolierten Lungen alternierend mit verschiedenen inspiratorischen Sauerstoffkonzentrationen ventiliert. Hierbei zeigte sich ein Minimum des ESR-Signals bei 2,5 % O2. Durch Zugabe von Superoxiddismutase (SOD) konnte bei allen Sauerstoffkonzentrationen die ESR-Signalintensität signifikant gesenkt werden. Dies deutet darauf hin, dass ein Anteil des ESR-Signals durch intravaskulär gebildetes Superoxid zustande kommt. Weiterhin kam es unter der Applikation von Phorbolmyristatacetat (PMA) zu einer Vasokonstriktion und zu einem signifikanten Anstieg der CPH-Oxidation. Unter dem Einsatz der SOD ließ sich die PMA induzierte CP˙-Bildung und die Vasokonstriktion komplett hemmen. Zur Untersuchung der Sauerstoffabhängigkeit der PMA induzierten Superoxidbildung wurden mit unterschiedlichen inspiratorischen Sauerstoffkonzentrationen ventiliert. Hierbei zeigte sich die höchste ESR-Signalintensität und die stärkste vasokonstriktive Antwort bei einer inspiratorischen Ventilation mit 5,0% O2. In neuesten Untersuchungen konnte diese PMA induzierte Vasokonstriktion mit Hilfe eines NADPH-Oxidase Inhibitors gehemmt werden. Die Daten zeigen, dass die durch PMA induzierte Vasokonstriktion wahrscheinlich durch Superoxid, generiert von NADPH-Oxidasen, vermittelt wird und dass es bei Hypoxie zu einer paradoxen, vermehrten O2.¯ Freisetzung kommen kann. Die Daten lassen weiterhin den Schluss zu, dass in der hypoxieabhängigen Regulation des pulmonalen Gefäßtonuses NADPH-Oxidasen involviert sein könnten. Weiterführende Untersuchungen sollten auf die zelluläre Ebene fokussieren, um die subzelluläre Quelle der hypoxieabhängigen ROS-Bildung zu identifizieren. Weiterhin kann die im Rahmen der vorliegenden Arbeit etablierte Methode zur Untersuchung der ROS-Bildung bei Ischämie/Reperfusion der Lungenstrombahn hilfreich sein.Reactive oxygen species (ROS) play an important role in biological systems. While it is largely accepted that ROS mediated oxidative damage occurs under pathophysiological conditions, recent evidence suggests a role for ROS as signalling molecules in physiological processes. In the respiratory system, ROS are thought to be involved in the pathogenesis of the adult respiratory distress syndrome (ARDS), sepsis and ischemia-reperfusion, but may also play a role in the development of hypoxia-and non-hypoxia-induced pulmonary hypertension. In the context of regulatory processes of the lung, a growing body of evidence is emerging indicating that ROS may contribute to signalling events such as underlying hypoxic pulmonary vasoconstriction (HPV), an essential mechanism matching lung perfusion to ventilation in order to optimize pulmonary gas exchange. The question of whether ROS generation is decreased or paradoxically increased during alveolar hypoxia, and the sources from which ROS may be derived under these conditions, are controversial. In fact, evidence exists for both a decrease as well as increase in lung ROS generation during alveolar hypoxia. Both mitochondria and NADPH oxidases have been proposed as sources of ROS generation underlying HPV. In this thesis a method combining electronspinresonance (ESR) spectroscopy with the spin trapping technique for measurement of superoxide release from isolated perfused and ventilated rabbit lungs was established. Superoxide was detected by the cyclic hydroxylamine l-hydroxy-3-carboxy- 2,2,5,5-tetramethylpyrrolidine (CPH) which was recently introduced for quantitative ROS measurements in biological systems. ROS formation can be quantified by detection of the corresponding radical 3-carboxy-proxyl (CP.) using ESR spectroscopy. After having proven the feasibility of CPH for ROS detection in the isolated perfused and ventilated rabbit lungs, the autooxidation of CPH was characterized. Perfusing lungs with CPH over a range of different inspired oxygen concentrations revealed an oxygen-dependence of CP. formation rate, with a minimum at 2,5 % O2. Addition of superoxide dismutase (SOD) to the buffer fluid illustrated that a portion of this intravascular ROS release was attributable to superoxide. Intravascular stimulation of the lungs by phorbolmyristateacetate (PMA) caused a rapid increase in CP. formation, concomitant with pulmonary vasoconstriction. Both the PMA-induced CPH oxidation and the vasoconstrictor response were completely suppressed by SOD. When the PMA challenge was performed at different oxygen concentrations, maximum superoxide liberation and pulmonary vasoconstriction occurred at 5,0 % O2. The PMA-induced vasoconstriction in the isolated lung experiments was inhibited by a NADPH-oxidase inhibitor These data demonstrate that the PMA-induced vasoconstriction is caused by superoxide generated from NADPH oxidases and that superoxide release may paradoxical be increased in hypoxia. NADPH oxidases thus may contribute to the hypoxia-dependent regulation of pulmonary vascular tone. Further experiments should be focused on cellular and subcellular sources of the hypoxia-dependent ROS-generation. Furthermore the established method may be helpful to clarify the role of ROS-generation in ischemia/reperfusion in the pulmonary vasculature

Endocannabinoid 2-arachidonoylglycerol is elevated in the coronary circulation during acute coronary syndrome.

OBJECTIVES:The endocannabinoid system modulates coronary circulatory function and atherogenesis. The two major endocannabinoids (eCB), 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamide (AEA), are increased in venous blood from patients with coronary artery disease (CAD). However, given their short half-life and their autocrine/paracrine mechanism of action, eCB levels in venous blood samples might not reflect arterial or coronary eCB concentrations. The aim of this cross-sectional study was to identify the local concentration profile of eCB and to detect whether and how this concentration profile changes in CAD and NSTEMI versus patients without CAD. METHODS AND RESULTS:83 patients undergoing coronary angiography were included in this study. Patients were divided into three groups based on their definite diagnosis of a) no CAD, b) stable CAD, or c) non-ST-segment elevation myocardial infarction (NSTEMI). Blood was drawn from the arterial sheath and the aorta in all patients and additionally distal to the culprit coronary lesion in CAD- and NSTEMI patients. 2-AG levels varied significantly between patient groups and between the sites of blood extraction. The lowest levels were detected in patients without CAD; the highest 2-AG concentrations were detected in NSTEMI patients and in the coronary arteries. Peripheral 2-AG levels were significantly higher in NSTEMI patients (107.4 ± 28.4 pmol/ml) than in CAD- (17.4 ± 5.4 pmol/ml; p < 0.001), or no-CAD patients (23.9 ± 7.1 pmol/ml; p < 0.001). Moreover, coronary 2-AG levels were significantly higher in NSTEMI patients than in CAD patients (369.3 ± 57.2 pmol/ml vs. 240.1 ± 25.3 pmol/ml; p = 0.024). CONCLUSIONS:2-AG showed significant variability in arterial blood samples drawn from distinct locations. Possibly, lesional macrophages synthesise 2-AG locally, which thereby contributes to endothelial dysfunction and local inflammation

The modified MIDA-Score predicts mid-term outcomes after interventional therapy of functional mitral regurgitation.

AimsThe preprocedural assessment of outcomes and patients' prognosis after interventional therapy of functional MR (FMR) is uncertain. Therefore, we aim to develop an easy-to-handle scoring system for adequate prediction of individual outcomes in patients with FMR after the interventional treatment.Materials and methodsWe retrospectively used medical data of patients with symptomatic FMR, who underwent transcatheter mitral valve repair (TMVR) from January 2014 to August 2016 in our heart center. All patients had the mean follow-up of 18 months. All clinical and echocardiographic data originate from the "Bonner Mitral Valve Register Database".ResultsWe included 105 patients (76,7±8,8 years, 50,6% female) with symptomatic (NYHA functional class>II) moderate-to-severe or severe FMR at surgical high-risk. We modified the MIDA-Score for degenerative MR (DMR) according to the varying underlying pathomechanisms of FMR, called as "The modified MIDA Score". We found all-cause mortality of 7% within 18 months after the procedure. 34,1% of our cohort was rehospitalized; 90% of those were due to cardiovascular causes. The modified MIDA score was found to be a strong predictor for mortality and rehospitalization in patients with FMR (AUC: 0,89) and superior to the other conventional scoring systems in prediction of mortality (The modified MIDA-Score: AUC: 0,8, EuroSCORE II: AUC: 0,57, STS-Score: AUC: 0,51). The logistic regression analysis showed the modified MIDA score > 9 points to be the strongest predictor for mortality and rehospitalization after TMVR (OR: 3,35, p = 0,011).ConclusionThe modified MIDA score was found to be a promising, easy-to-handle, elementary scoring system for adequate prediction of individual postinterventional prognosis in patients with FMR undergoing TMVR. Further evaluation and validation of this novel scoring system in prospective multicentric studies with a large number of patients is warranted

Comparative study of pressure (ankle-brachial pressure index) and flow (strain gauge plethysmography and reactive hyperaemia) measurements in diagnosis of peripheral arterial disease in patients with severe aortic stenosis.

BackgroundThe measurement of the ankle-brachial pressure index is a straightforward method for the detection of peripheral disease in the lower limbs. Only a few old studies with small numbers of patients have been conducted comparing the gold standard, ankle-brachial pressure index measurement, with strain gauge plethysmography and reactive hyperaemia for detecting peripheral arterial disease. The purpose of this study was to evaluate the feasibility and accuracy of strain gauge plethysmography values compared with the Doppler ultrasound method, ankle-brachial pressure index, in the assessment of peripheral arterial disease, especially in patients with severe aortic stenosis.Methods221 ankle-brachial pressure index measurements and strain gauge plethysmography measurements of patients with suspected peripheral arterial disease, diagnosed peripheral arterial disease with or without aortic stenosis were compared.ResultsIrrespective of aortic stenosis in patients with and without peripheral arterial disease, the resting arterial blood flow was within the normal range. In patients with aortic stenosis, the time-to-peak flow couldn't detect peripheral arterial disease and was found to be a false negative. In patients without aortic stenosis, time-to-peak flow correlated well with the ankle-brachial pressure index for detecting peripheral arterial disease. Peak flow at 5 seconds was the one of the flow values that correlated with ankle-brachial pressure index and detected peripheral arterial disease in patients with and without aortic stenosis.ConclusionPeak flow at 5 seconds is one of flow value that correlated well with ankle-brachial pressure index in detecting peripheral arterial disease in patients with and without aortic stenosis. Detection of peripheral arterial disease in patients with severe aortic stenosis seems to be less sensitive with flow measurements than with ankle-brachial pressure index

CHA2DS2-VASC score predicts coronary artery disease progression and mortality after ventricular arrhythmia in patients with implantable cardioverter-defibrillator

Aim: The CHA2DS2-VASC score has expanded its use beyond the initial purpose of predicting the risk of stroke in patients with atrial fibrillation. We aimed to investigate the value of the CHA2DS2-VASC score as a risk assessment tool to predict relevant coronary artery disease (CAD) leading to percutaneous coronary intervention (PCI), and all-cause mortality after detected ventricular arrhythmia (VA) in patients with an Implantable Cardioverter-Defibrillator (ICD). Methods: A total of 183 ICD-patients who underwent coronary angiography after VA were included and classified according to their CHA2DS2-VASC score in a low(1-3), intermediate(4-5) and high(6-8) score group. We evaluated the predictive value of CHA2DS2-VASC score for the presence of relevant CAD leading to percutaneous coronary intervention (PCI), as well as late all-cause mortality. Results: A total of 60 patients (32.8%) had significant CAD and underwent successful PCI. After adjustment for relevant parameters such as ischemic cardiomyopathy, angina pectoris, left ventricular ejection fraction, CHA2DS2-VASC score remained the only independent predictor of CAD leading to PCI [HR 1.73 (1.07–2.80)]. The Area under curve was 0.64 (0.56–72, p = 0.002). Kaplan-Meier analysis and log-rank showed an increased three-year mortality of ICD-patients with an intermediate or high score after VA (p = 0.003). Multivariate cox-regression analysis revealed that CHA2DS2-VASC score was also independently associated with all-cause mortality following adjustment for clinically relevant variables (HR 2.20, 1.17–4.14). Conclusions: CHA2DS2-VASC score can be a predictor of CAD leading to PCI in ICD-patients after VA. ICD-Patients with a high score have an increased risk for reduced three-year all-cause mortality after VA

Impact of peroxisome proliferator-activated receptor γ on angiotensin II type 1 receptor-mediated insulin sensitivity, vascular inflammation and atherogenesis in hypercholesterolemic mice

INTRODUCTION: The angiotensin II type 1 receptor (AT1R) and the peroxisome proliferator-activated receptor γ (PPARγ) have been implicated in the pathogenesis of atherosclerosis. A number of studies have reported that AT1R inhibition or genetic AT1R disruption and PPARγ activation inhibit vascular inflammation and improve glucose and lipid metabolism, underscoring a molecular interaction of AT1R and PPARγ. We here analyzed the hypothesis that vasculoprotective anti-inflammatory and metabolic effects of AT1R inhibition are mediated by PPARγ. MATERIAL AND METHODS: Female ApoE(–/–)/AT1R(–/–) mice were fedwith a high-fat and cholesterol-rich diet and received continuous treatment with the selective PPARγ antagonist GW9662 or vehicle at a rate of 700 ng/kg/min for 4 weeks using subcutaneously implanted osmotic mini-pumps. Additionally, one group of female ApoE(–/–) mice served as a control group. After treatment for 4 weeks mice were sacrificed and read-outs (plaque development, vascular inflammation and insulinsensitivity) were performed. RESULTS: Using AT1R deficient ApoE(–/–) mice (ApoE(–/–)/AT1R(–/–) mice) we found decreased cholesterol-induced endothelial dysfunction and atherogenesis compared to ApoE(–/–) mice. Inhibition of PPARγ by application of the specific PPARγ antagonist GW9662 significantly abolished the anti-atherogenic effects of AT1R deficiency in ApoE(–/–)/AT1R(–/–) mice (plaque area as % of control: ApoE(–/–): 39 ±5%; ApoE(–/–)/AT1R(–/–): 17 ±7%, p = 0.044 vs. ApoE(–/–); ApoE(–/–)/AT1R(–/–) + GW9662: 31 ±8%, p = 0.047 vs. ApoE(–/–)/AT1R(–/–)). Focusing on IL6 as a pro-inflammatory humoral marker we detected significantly increased IL-6 levels in GW9662-treated animals (IL-6 in pg/ml: ApoE(–/–): 230 ±16; ApoE(–/–)/AT1R(–/–): 117 ±20, p = 0.01 vs. ApoE(–/–); ApoE(–/–)/AT1R(–/–) + GW9662: 199 ±20, p = 0.01 vs. ApoE(–/–)/AT1R(–/–)), while the anti-inflammatory marker IL-10 was significantly reduced after PPARγ inhibition in GW9662 animals (IL-10 in pg/ml: ApoE(–/–): 18 ±4; ApoE(–/–)/AT1R(–/–): 55 ±12, p = 0.03 vs. ApoE(–/–); ApoE(–/–)/AT1R(–/–) + GW9662: 19 ±4, p = 0.03 vs. ApoE(–/–)/AT1R(–/–)). Metabolic parameters of glucose homeostasis (glucose and insulin tolerance test) were significantly deteriorated in ApoE(–/–)/AT1R(–/–) mice treated with GW9662 as compared to vehicle-treated ApoE(–/–)/AT1R(–/–) mice. Systolic blood pressure and plasma cholesterol levels were similar in all groups. CONCLUSIONS: Genetic disruption of the AT1R attenuates atherosclerosis and improves endothelial function in an ApoE(–/–) mouse model of hypercholesterolemia-induced atherosclerosis via PPARγ, indicating a significant role of PPARγ in reduced vascular inflammation, improvement of insulin sensitivity and atheroprotection of AT1R deficiency