Gregor Muller

NO‐mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells

The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (·O2−) formation. In this study, we analysed the effect of laminar shear stress on ·O2− formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription‐polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone‐and‐plate viscometer for up to 24 h. Short‐term application of shear stress transiently induced ·O2− formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds‐tat, but NAD(P)H oxidase subunit expression was unchanged. Long‐term arterial laminar shear stress (30 dyne cm−2, 24 h) down‐regulated ·O2− formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91phox and p47phox. In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down‐regulation of ·O2− formation, gp91phox and p47phox expression by long‐term laminar shear stress was blocked by l‐NAME. NO donor DETA‐NO down‐regulates ·O2− formation, gp91phox and p47phox expression in static cultures. In conclusion, our data suggest a transient activation of ·O2− formation by short‐term shear stress, followed by a down‐regulation of endothelial NAD(P)H oxidase in response to long‐term laminar shear stress. NO‐mediated down‐regulation by shear stress preferentially affects the gp91phox/p47phox‐containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/·O2− balance and the vasoprotective potential of physiological levels of laminar shear stress.

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22(phox) expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-beta no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22(phox). The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.

Impaired vascular function in small resistance arteries of LOX-1 overexpressing mice on high-fat diet

AIMS LOX-1 is a major vascular receptor for oxidized low-density lipoprotein (oxLDL). In this study, we analysed the impact of LOX-1 overexpression and high dietary fat intake on vascular function in small resistance arteries. METHODS AND RESULTS Relaxation of mesenteric arteries was measured using a wire myograph. Compared with the control group, mice overexpressing LOX-1 on a high-fat diet (FD) had preserved vascular smooth muscle relaxation, but impaired endothelium-dependent relaxation via NO. Vascular NO availability was decreased by exaggerated formation of reactive oxygen species and decreased endothelial NO synthase expression. Endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation via cytochrome P450 metabolites was increased in LOX-1 + FD animals, but did not completely compensate for the loss of NO. Currents of calcium-activated potassium channels with large conductance (BKCa channels) were measured by the voltage-clamp method. The BKCa current amplitudes were not altered in endothelial cells, but highly increased in vascular smooth muscle cells from resistance arteries of LOX-1-overexpressing mice on FD. BK(Ca) currents were activated by low-dose H2O2 and cytochrome P450 metabolites 11,12-EET and 14,15-EET as EDHF in control mice. CONCLUSION LOX-1 overexpression and FD caused functional changes in endothelial and vascular smooth muscle cells of small resistance arteries.

NAD(P)H Oxidase and Endothelial Dysfunction

The regulation of endothelial function plays an important role in the development and progression of metabolic and cardiovascular diseases. A critical determinant of endothelial function is the balance between nitric oxide and reactive oxygen species. Endothelium-derived NO availability can be limited by enhanced formation of reactive oxygen species. Major sources of reactive oxygen species in the vessel wall are NAD(P)H oxidase complexes. This review summarizes the impact of vascular NAD(P)H oxidase-derived reactive oxygen species on atherosclerosis and endothelial dysfunction. Changes in NAD(P)H oxidase expression and activity have clinical implications. Mutations in NAD(P)H oxidase subunits can lead to impaired oxidative burst in leukocytes and chronic granulomatous disease. In contrast, normalization of increased expression and activity of NAD(P)H oxidase in endothelial dysfunction and vascular disorders can be considered as a novel therapeutic strategy in the treatment of cardiovascular diseases.

Investigation of murine Vasodynamics by Fourier Domain Optical Coherence Tomography

In vivo imaging of blood vessels obtain useful insights in characterizing the dynamics of vasoconstriction and vasodilation. Fourier domain optical Coherence Tomography (FD-OCT) imaging technique permits in vivo investigation of blood vessels in their anatomical context without preparation traumata by temporal resolved image stacks. OCT is an optical, contact less imaging technique based on Michelson interferometry of short coherent near infrared light. Particularly by the possibility of a contact-less measurement and the high axial resolution up to 10 microns OCT is superior to an investigation by ultra sound measurement. Furthermore we obtain a high time resolution of vessel dynamic measurements with the used Fourier domain OCT-system by a high A-scan rate [1,22kHz]. In this study the model of saphenous artery was chosen for analyzing function and dynamics. The arteria saphena in the mouse is a suitable blood vessel due to the small inner diameter, a sensitive response to vasoactive stimuli and an advantageous anatomically position. Male wild type mice (C57BL/6) at the age of 8 weeks were fed control or high-fat diet for 10 weeks before analyzing the vasodynamics. The blood vessel was stimulated by dermal application of potassium to induce vasoconstriction or Sodium-Nitroprusside (SNP) to induce vasodilation. The morphology of the a. saphena and vein was determined by 3D image stacks. Time series (72 seconds, 300x512 pixel per frame) of cross-sectional images were analysed using semi automatic image processing software. Time course of dynamic parameters of the vessel was measured.

In-vivo Fourier domain optical coherence tomography as a new tool for investigation of vasodynamics in the mouse model

In-vivo imaging of the vascular system can provide novel insight into the dynamics of vasoconstriction and vasodilation. Fourier domain optical coherence tomography (FD-OCT) is an optical, noncontact imaging technique based on interferometry of short-coherent near-infrared light with axial resolution of less than 10 microm. In this study, we apply FD-OCT as an in-vivo imaging technique to investigate blood vessels in their anatomical context using temporally resolved image stacks. Our chosen model system is the murine saphenous artery and vein, due to their small inner vessel diameters, sensitive response to vasoactive stimuli, and advantageous anatomical position. The vascular function of male wild-type mice (C57BL/6) is determined at the ages of 6 and 20 weeks. Vasoconstriction is analyzed in response to dermal application of potassium (K(+)), and vasodilation in response to sodium nitroprusside (SNP). Vasodynamics are quantified from time series (75 sec, 4 frames per sec, 330 x 512 pixels per frame) of cross sectional images that are analyzed by semiautomated image processing software. The morphology of the saphenous artery and vein is determined by 3-D image stacks of 512 x 512 x 512 pixels. Using the FD-OCT technique, we are able to demonstrate age-dependent differences in vascular function and vasodynamics.

An elevated lipoprotein(a) plasma level as a cardiovascular risk factor

INTRODUCTION The causal association of elevated lipoprotein(a) (Lp(a)) plasma levels with the increased cardiovascular risk is still controversial and presently there are no standard recommendations on managing of hyperLp(a)emia. Our retrospective analysis is aimed to explore the Lp(a) thresholds, the magnitude of various cardiovascular risk factors and their combinations. METHODS The files of 544 outpatients from our Outpatient Department of Lipid Disorders were divided into quintiles with respect to Lp(a) levels and reviewed regarding age, gender, Body Mass Index, dyslipidemias, arterial hypertension, diabetes mellitus, smoking and incidence of vascular events in coronaries, carotids and lower extremities. Furthermore we built 15 small quantiles to identify the Lp(a) threshold more precisely. RESULTS The incidence odds ratio for cardiovascular events rose from 2.65 in the 2nd quintile with Lp(a) 483-821 mg/L to 6.36 in the 5th quintile (Lp(a) ≥ 1495 mg/L). The relative risk of cardiovascular events was 0.08 in subjects with a Lp(a) level under 232 mg/l and 3.6 at Lp(a) ≥ 315 mg/L. The magnitude of the combination of elevated Lp (a) with arterial hypertension factor exceeded that of gender, age and combination of arterial hypertension with smoking. CONCLUSIONS A Lp(a) plasma level of higher than about 300 mg/L seems to be a threshold for occurring of cardiovascular events. The combination of raised Lp(a) with arterial hypertension was found to be the most important cardiovascular risk factor. Lp(a) levels under 232 mg/L appeared to be a marker for good prognosis.

Analysis of murine vascular function in vivo by optical coherence tomography in response to high-fat diet.

In this study, we demonstrate the application of optical coherence tomography (OCT) as a contactless imaging technique to analyze vasodynamics in small blood vessels in vivo. The transluminal OCT imaging of vessels avoids micro traumata in the endothelium and circumvents surgical intervention. It can be performed in the intact perfused vessel and provides a new method to measure vascular function and dynamics in vivo. The resolution of 10 mum and the velocity of image acquisition are adequate to detect differences in the inner diameter, the maximal velocity, or the time to half-maximal diameter change of small vessels. We applied this new technology to study the vascular dynamics in small vessels of 6- and 20-week-old C57BL/6 mice in vivo. In addition, we determined by this technique the impact of a high-fat diet for 14 weeks on vascular function in 20-week-old animals. The diameter of the saphenous artery was increased under resting conditions, after vasoconstriction and after vasodilatation in 20-week-old animals on normal chow and high-fat diet, compared to 6-week-old animals. High-fat diet caused a significantly impaired vasoconstriction in the saphenous artery. The maximal velocity of diameter changes of the saphenous artery was determined by time-resolved OCT imaging. A significant reduction of this parameter was found during vasoconstriction in 20-week-old mice on high-fat diet, compared to 6-week-old animals. In conclusion, transluminal optical coherence tomography imaging is a novel and useful technique to analyze the impaired vasodynamics of small arteries in response to high-fat diet in vivo.

Quantifizierung von Flussgeschwindigkeiten mit dem Verfahren der Fourier Domain Optische KohärenztomografieFlow Velocity Quantification with Fourier Domain Optical Coherence Tomography

Zusammenfassung In dieser Studie werden zwei Methoden der Flussmessung, basierend auf dem Verfahren der Fourier Domain Optische Kohärenztomografie (FD OCT) vorgestellt. Das erste Verfahren beruht auf der Analyse der Phasenverschiebung zwischen konsekutiven Tiefensignalen durch axial bewegte Strukturen. Mit der zweiten Methode besteht die Möglichkeit höhere Geschwindigkeiten über den Signalabfall, hervorgerufen durch schnell bewegte Streuzentren, zu bestimmen.

Vasodynamics of the Murine Arteria Saphena by Optical Coherence Tomography

To investigate the pathogenesis of arteriosclerosis and its negative effects on the vasodynamics, in vitro experiments on isolated blood vessels as well as in vivo experiments are essential. The isometric force measurement commonly used to analyse the vasoconstriction of isolated blood vessels can not be applied in the in vivo situation. In contrast Optical Coherence Tomography (OCT) allows the acquisition of 2D cross sectional images und 3D cross sectional image stacks of vessel sections. We demonstrate that our two Fourier domain OCT-systems with their automated image analysis are suitable investigative tools for studying the stage of arteriosclerotic plaque formation as well as for examining the response of the blood vessel to vasomotor stimuli in the mouse model.