Alexander Scholz

Glucose-independent improvement of vascular dysfunction in experimental sepsis by dipeptidyl-peptidase 4 inhibition

AIMS Dipeptidyl peptidase-4 (DPP-4) inhibitors are a novel class of drugs for the treatment of hyperglycaemia. Preliminary evidence suggests that their antioxidant and anti-inflammatory effects may have beneficial effects on the cardiovascular complications of diabetes. In the present study, we investigate in an experimental sepsis model whether linagliptin exerts pleiotropic vascular effects independent of its glucose-lowering properties. METHODS AND RESULTS Linagliptin (83 mg/kg chow for 7 days) was administered in a rat model of lipopolysaccharide (LPS) (10 mg/kg, single i.p. dose/24 h)-induced sepsis. Vascular relaxation, reactive oxygen species (ROS) formation, expression of NADPH oxidase subunits and proinflammatory markers, and white blood cell infiltration in the vasculature were determined. Oxidative burst and adhesion of isolated human neutrophils to endothelial cells were measured in the presence of different DPP-4 inhibitors, and their direct vasodilatory effects (isometric tension in isolated aortic rings) were compared. In vivo linagliptin treatment ameliorated LPS-induced endothelial dysfunction and was associated with reduced formation of vascular, cardiac, and blood ROS, aortic expression of inflammatory genes and NADPH oxidase subunits in addition to reduced aortic infiltration with inflammatory cells. Linagliptin was the most potent inhibitor of oxidative burst in isolated activated human neutrophils and it suppressed their adhesion to activated endothelial cells. Of the inhibitors tested, linagliptin and alogliptin had the most pronounced direct vasodilatory potency. CONCLUSION Linagliptin demonstrated pleiotropic vasodilatory, antioxidant, and anti-inflammatory properties independent of its glucose-lowering properties. These pleiotropic properties are generally not shared by other DPP-4 inhibitors and might translate into cardiovascular benefits in diabetic patients.

Molecular Mechanisms of the Crosstalk Between Mitochondria and NADPH Oxidase Through Reactive Oxygen Species—Studies in White Blood Cells and in Animal Models

AIMS Oxidative stress is involved in the development of cardiovascular disease. There is a growing body of evidence for a crosstalk between different enzymatic sources of oxidative stress. With the present study, we sought to determine the underlying crosstalk mechanisms, the role of the mitochondrial permeability transition pore (mPTP), and its link to endothelial dysfunction. RESULTS NADPH oxidase (Nox) activation (oxidative burst and translocation of cytosolic Nox subunits) was observed in response to mitochondrial reactive oxygen species (mtROS) formation in human leukocytes. In vitro, mtROS-induced Nox activation was prevented by inhibitors of the mPTP, protein kinase C, tyrosine kinase cSrc, Nox itself, or an intracellular calcium chelator and was absent in leukocytes with p47phox deficiency (regulates Nox2) or with cyclophilin D deficiency (regulates mPTP). In contrast, the crosstalk in leukocytes was amplified by mitochondrial superoxide dismutase (type 2) (MnSOD(+/-)) deficiency. In vivo, increases in blood pressure, degree of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) dysregulation/uncoupling (e.g., eNOS S-glutathionylation) or Nox activity, p47phox phosphorylation in response to angiotensin-II (AT-II) in vivo treatment, or the aging process were more pronounced in MnSOD(+/-) mice as compared with untreated controls and improved by mPTP inhibition by cyclophilin D deficiency or sanglifehrin A therapy. INNOVATION These results provide new mechanistic insights into what extent mtROS trigger Nox activation in phagocytes and cardiovascular tissue, leading to endothelial dysfunction. CONCLUSIONS Our data show that mtROS trigger the activation of phagocytic and cardiovascular NADPH oxidases, which may have fundamental implications for immune cell activation and development of AT-II-induced hypertension.

Nitroglycerin-Induced Endothelial Dysfunction and Tolerance Involve Adverse Phosphorylation and S-Glutathionylation of Endothelial Nitric Oxide Synthase

Objective—Continuous administration of nitroglycerin (GTN) causes tolerance and endothelial dysfunction by inducing reactive oxygen species (ROS) production from various enzymatic sources, such as mitochondria, NADPH oxidase, and an uncoupled endothelial nitric oxide synthase (eNOS). In the present study, we tested the effects of type 1 angiotensin (AT1)-receptor blockade with telmisartan on GTN-induced endothelial dysfunction in particular on eNOS phosphorylation and S-glutathionylation sites and the eNOS cofactor synthesizing enzyme GTP–cyclohydrolase I. Methods and Results—Wistar rats were treated with telmisartan (2.7 or 8 mg/kg per day PO for 10 days) and with GTN (50 mg/kg per day SC for 3 days). Aortic eNOS phosphorylation and S-glutathionylation were assessed using antibodies against phospho-Thr495 and Ser1177 or protein-bound glutathione, which regulate eNOS activity and eNOS-dependent superoxide production (uncoupling). Expression of mitochondrial aldehyde dehydrogenase was determined by Western blotting. Formation of aortic and cardiac ROS was assessed by fluorescence, chemiluminescence, and 3-nitrotyrosine/malondialdehyde-positive protein content. Telmisartan prevented endothelial dysfunction and partially improved nitrate tolerance. Vascular, cardiac, mitochondrial, and white blood cell ROS formation were significantly increased by GTN treatment and inhibited by telmisartan. GTN-induced decrease in Ser1177, increase in Thr495 phosphorylation or S-glutathionylation of eNOS, and decrease in mitochondrial aldehyde dehydrogenase expression were normalized by telmisartan. Conclusion—These data identify modification of eNOS phosphorylation as an important component of GTN-induced endothelial dysfunction. Via its pleiotropic “antioxidant” properties, telmisartan prevents, at least in part, GTN-induced oxidative stress, nitrate tolerance, and endothelial dysfunction.

Dynamic 19F-MRI of pulmonary ventilation using sulfur hexafluoride (SF6) gas

A new method for dynamic imaging of pulmonary wash‐in and wash‐out kinetics of inhaled sulfur hexafluoride (SF6) gas was developed. Measurements at the fluorine‐19 Larmor frequency were performed in pigs using a gradient echo pulse sequence with 0.5 ms echo time and a measurement time of 9.1 s per image. Dynamic MRI was performed during wash‐in and wash‐out of SF6 gas in mechanically ventilated porcine lungs. A postprocessing strategy was developed for quantitative determination of wash‐out time constants in the presence of noise. Mean wash‐out constants were 4.78 ± 0.48 breaths vs. 4.33 ± 0.76 breaths for left and right lung when ventilation was performed with low tidal volume, and 1.73 ± 0.16 breaths vs. 1.85 ± 0.11 breaths with high tidal volume ventilation. In conclusion, breath‐hold MRI of SF6 gas is feasible in large animals. Moreover, regional wash‐in and wash‐out kinetics of SF6 can be determined noninvasively with this new method. Potential human applications are discussed. Magn Reson Med 45:605–613, 2001.

Lung ventilation- and perfusion-weighted Fourier decomposition magnetic resonance imaging: In vivo validation with hyperpolarized 3He and dynamic contrast-enhanced MRI

The purpose of this work was to validate ventilation‐weighted (VW) and perfusion‐weighted (QW) Fourier decomposition (FD) magnetic resonance imaging (MRI) with hyperpolarized 3He MRI and dynamic contrast‐enhanced perfusion (DCE) MRI in a controlled animal experiment. Three healthy pigs were studied on 1.5‐T MR scanner. For FD MRI, the VW and QW images were obtained by postprocessing of time‐resolved lung image sets. DCE acquisitions were performed immediately after contrast agent injection. 3He MRI data were acquired following the administration of hyperpolarized helium and nitrogen mixture. After baseline MR scans, pulmonary embolism was artificially produced. FD MRI and DCE MRI perfusion measurements were repeated. Subsequently, atelectasis and air trapping were induced, which followed with FD MRI and 3He MRI ventilation measurements. Distributions of signal intensities in healthy and pathologic lung tissue were compared by statistical analysis. Images acquired using FD, 3He, and DCE MRI in all animals before the interventional procedure showed homogeneous ventilation and perfusion. Functional defects were detected by all MRI techniques at identical anatomical locations. Signal intensity in VW and QW images was significantly lower in pathological than in healthy lung parenchyma. The study has shown usefulness of FD MRI as an alternative, noninvasive, and easily implementable technique for the assessment of acute changes in lung function. Magn Reson Med, 2013.

19F-MRI of perflubron for measurement of oxygen partial pressure in porcine lungs during partial liquid ventilation.

A method for in vivo measurement of oxygen partial pressure (pO2) in porcine lungs during partial liquid ventilation (PLV) with perflubron (PFOB) was developed. A pulse sequence for high‐resolution MRI of the distribution of PFOB in the lung after intratracheal administration was developed as well. Moreover, quantitative measurements of longitudinal relaxation time T1 of 19F resonances for assessment of regional pO2 are described. Due to the need to acquire data during a single expiratory breathhold, only low SNRs were achieved in vivo. Therefore, simulations were performed to investigate the influence of background noise on T1 values calculated from data with low SNR. Based on these simulations, a postprocessing strategy was developed to correct for systematic errors by background noise prior to quantitative analysis. Results of a pilot study in pigs under conditions of PLV are presented. Magn Reson Med 47:82–89, 2002.

Hyperglycemia and oxidative stress in cultured endothelial cells - a comparison of primary endothelial cells with an immortalized endothelial cell line

Diabetes mellitus is a major risk factor for the development of cardiovascular disease and oxidative stress plays an important role in this process. Therefore, we investigated the effects of hyperglycemia on the formation of reactive oxygen species (ROS) and nitric oxide/cGMP signaling in two different endothelial cell cultures. Human umbilical vein endothelial cells (HUVEC) and EA.hy 926 cells showed increased oxidative stress and impaired NO-cGMP signaling in response to hyperglycemia. The major difference between the two different cell types was the dramatic decrease in viability in HUVEC whereas EA.hy cells showed rather increased growth under hyperglycemic conditions. Starvation led to an additional substantial decrease in viability and increased superoxide formation in HUVEC. Both endothelial cell types, HUVEC and EA.hy 926, may be used as models for vascular hyperglycemia. However, high growth medium should be used to avoid starvation-induced oxidative stress and cell death.

Subsecond fluorine-19 MRI of the lung

Minimal scan times in rapid fluorine‐19 MRI using sulfur hexafluoride (SF6) have been on the order of 10 s. Because of the very short T1 relaxation time of SF6 (T1 = 1.65 ms), high receiver bandwidths are necessary to allow for a high number of excitations. Since high bandwidths cause high levels of electronic noise, SNR per acquisition has been too low to further reduce scan time. The purpose of this study was to investigate whether scan times could be reduced using hexafluoroethane (C2F6), a gas with a longer T1 (T1 = 7.9 ms) at a relatively low bandwidth of 488 Hz/pixel. Gradient‐echo images were acquired during and after completion of the wash‐in of a 70% C2F6‐30% O2 mixture. Peak SNR values of 16 and 7.9 were observed for coronal projection images acquired within 2 s and 260 ms, respectively. These results demonstrate that subsecond imaging is feasible using C2F6. Magn Reson Med, 2006.

Magnetic resonance imaging of dissolved hyperpolarized 129Xe using a membrane-based continuous flow system.

A technique for continuous production of solutions containing hyperpolarized (129)Xe is explored for MRI applications. The method is based on hollow fiber membranes which inhibit the formation of foams and bubbles. A systematic analysis of various carrier agents for hyperpolarized (129)Xe has been carried out, which are applicable as contrast agents for in vivo MRI. The image quality of different hyperpolarized Xe solutions is compared and MRI results obtained in a clinical as well as in a nonclinical MRI setting are provided. Moreover, we demonstrate the application of (129)Xe contrast agents produced with our dissolution method for lung MRI by imaging hyperpolarized (129)Xe that has been both dissolved in and outgassed from a carrier liquid in a lung phantom, illustrating its potential for the measurement of lung perfusion and ventilation.

Controlling diffusion of He-3 by buffer gases: A structural contrast agent in lung MRI

To study the influence of admixing inert buffer gases to laser‐polarized 3He in terms of resulting diffusion coefficients and the consequences for image contrast and resolution.