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

"Possible TRALI" developed during bilateral total knee arthroplasty replacement -A case report-

Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related morbidity and mortality. However, it is frequently not diagnosed and under-reported, which could result in inappropriate treatment. Diagnostic definition for TRALI consists of hypoxia and bilateral pulmonary edema occurring during or within 6 hours of a transfusion in the absence of cardiac failure or intravascular volume overload. Here, we report a fatal case, which resulted from under-recognition and misdiagnosis of TRALI occurring during transfusion with packed red blood cells during a bilateral total knee replacement

Procedural Outcomes of a Self-Expanding Transcatheter Heart Valve in Patients with Porcelain Aorta

Background: Severe calcification of the ascending aorta increases the peri-operative risk for neurological complications in patients with severe aortic stenosis. Transcatheter aortic valve implantation (TAVI) seems to be an optimal treatment option in these patients. However, the impact of the extent of aortic calcification on procedural and neurological outcomes during TAVI is unclear. Methods: Data from 3010 patients with severe native aortic valve stenosis treated with ACURATE neo/neo2 from May 2012 to July 2022 were evaluated and matched by 2-to-1 nearest-neighbor matching to identify one patient with porcelain aorta (PA) (n = 492) compared with two patients without PA (n = 984). PA was additionally subdivided into circumferential (classic PA) (n = 89; 3.0%) and non-circumferential (partial PA) (n = 403; 13.4%) calcification. We compared outcomes according to VARC-3 criteria among patients with and without PA and identified predictors for occurrence of stroke in the overall population. Results: Technical success (88.5% vs. 87.4%, p = 0.589) and device success at 30 days (82.3% vs. 81.5%, p = 0.755) after transcatheter ACURATE neo/neo2 implantation according to VARC-3 definition was high and did not differ between non-calcified aortas or PA. The rate of in-hospital complications according to VARC-3-definitions was low in both groups. Rates of all stroke (3.2% (n = 31) vs. 2.6% (n = 13), p = 0.705) or transitory ischemic attacks (1.1% vs. 1.2%, p = 1.000) did not differ significantly. Thirty-day all-cause mortality did not differ (3.0% vs. 3.2%, RR 1.1; p = 0.775). Overall device migration/embolization (OR 5.0 [2.10;11.87]), severe bleeding (OR 1.79 [1.11;2.89]), and major structural cardiac complications (OR 3.37 [1.32;8.57]) were identified as independent predictors for in-hospital stroke in a multivariate analysis after implantation of ACURATE neo/neo2. Conclusion: A porcelain aorta does not increase the risk of neurological complications after transfemoral ACURATE neo/neo2 implantation. Based on these findings, transfemoral ACURATE neo/neo2 implantation is safe in these particularly vulnerable patients

Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial Nox2 in vivo. To address this question, we generated transgenic mice with endothelial-specific overexpression of Nox2 (Tg) and studied the effects on endothelial function and blood pressure. Tg had an about twofold increase in endothelial Nox2 levels which was accompanied by an increase in p22phox levels but no change in levels of other Nox isoforms or endothelial nitric oxide synthase (eNOS). Basal NADPH oxidase activity, endothelial function and blood pressure were unaltered in Tg compared to wild-type littermates. Angiotensin II caused a greater increase in ROS production in Tg compared to wild-type aorta and attenuated acetylcholine-induced vasorelaxation. Both low and high dose chronic angiotensin II infusion increased telemetric ambulatory blood pressure more in Tg compared to wild-type, but with different patterns of BP change and aortic remodeling depending upon the dose of angiotensin II dose. These results indicate that an increase in endothelial Nox2 levels contributes to angiotensin II-induced endothelial dysfunction, vascular remodeling and hypertension

NADPH oxidases in cardiovascular disease: insights from in vivo models and clinical studies

NADPH oxidase family enzymes (or NOXs) are the major sources of reactive oxygen species (ROS) that are implicated in the pathophysiology of many cardiovascular diseases. These enzymes appear to be especially important in the modulation of redox-sensitive signalling pathways that underlie key cellular functions such as growth, differentiation, migration and proliferation. Seven distinct members of the family have been identified of which four (namely NOX1, 2, 4 and 5) may have cardiovascular functions. In this article, we review our current understanding of the roles of NOX enzymes in several common cardiovascular disease states, with a focus on data from genetic studies and clinical data where available

Inflammation Resolution and the Induction of Granulocyte Apoptosis by Cyclin-Dependent Kinase Inhibitor Drugs

Inflammation is a necessary dynamic tissue response to injury or infection and it's resolution is essential to return tissue homeostasis and function. Defective or dysregulated inflammation resolution contributes significantly to the pathogenesis of many, often common and challenging to treat human conditions. The transition of inflammation to resolution is an active process, involving the clearance of inflammatory cells (granulocytes), a change of mediators and their receptors, and prevention of further inflammatory cell infiltration. This review focuses on the use of cyclin dependent kinase inhibitor drugs to pharmacologically target this inflammatory resolution switch, specifically through inducing granulocyte apoptosis and phagocytic clearance of apoptotic cells (efferocytosis). The key processes and pathways required for granulocyte apoptosis, recruitment of phagocytes and mechanisms of engulfment are discussed along with the cumulating evidence for cyclin dependent kinase inhibitor drugs as pro-resolution therapeutics

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