Effects of maternal PETN treatment of spontaneously hypertensive rats on blood pressure in the offspring

Pentaerithrityltetranitrat (PETN) ist ein organisches Nitrat und wird in der Klinik zur Behandlung der Angina Pectoris eingesetzt. PETN hat, wenn direkt verabreicht, kaum Wirkung auf den Blutdruck. Diese Arbeit wurde konzipiert, um einen potentiellen „perinatalen Programmierung“-Effekt von PETN in spontan-hypertensiven Ratten (SHR), einem Rattenmodel der genetischen Hypertonie, zu testen. Die F0-Elterntiere wurden mit PETN (50 mg/kg/Tag) während der Schwangerschaft und der Laktation behandelt; die F1-Nachkommen bekamen nach der Ablaktation normales Haltungsfutter. Der Blutdruck wurde an den Nachkommen vom 3. Monat bis zum 8. Monat nach der Geburt gemessen. Maternale PETN-Behandlung hatte kaum Wirkung auf den Blutdruck in den männlichen SHR-Nachkommen. Dagegen zeigten die weiblichen Nachkommen der PETN-Behandlungsgruppe eine persistente Reduktion des Blutdrucks. Der systolische Blutdruck war in den weiblichen Nachkommen in der PETN-Gruppe etwa 13 mmHg niedriger im 4. Monat und etwa 10 mmHg niedriger im 8. Monat als in den Kontrolltieren. Dieser lang-anhaltende Effekt ging mit einer substanziellen Änderung der Genexpression einher, die auch beim 8. Monat noch nachzuweisen war. In den Aorten der weiblichen F1-Nachkommen wurde Veränderungen an Genexpression der α-adrenergen Rezeptoren sowie Endothelin-Rezeptoren festgestellt, die aber funktionell von minimaler Bedeutung für die PETN-Wirkung waren. Hingegen war eine klare Rolle des StickstoffmoNOXid (NO) zu sehen. Maternale PETN-Behandlung führte zur Heraufregulation der endothelialen NO-Synthase (eNOS) und der GTP-Cyclohydrolase I (GCH-1). GCH-1 ist für die Biosynthese des Tetrahydrobiopterins, eines essentiellen eNOS-Kofaktors, entscheidend, und dadurch auch für die eNOS-Funktionalität. Zusätzlich wurden auch anti-oxidative Enzyme wie die mitochondriale Superoxid-Dismutase (SOD2), die Glutathion-Peroxidase 1 (GPx1) und die Hem-Oxygenase 1 (HO-1) heraufreguliert, und die Superoxid-produzierende NADPH-Oxidase NOX1 herunterreguliert. Dies kann zur Verminderung vom oxidativen Stress und Erhöhung der NO-Bioverfügbarkeit führen. Letztlich wurde auch ~ 74 ~ die Sirtuin 1 (SIRT1) durch maternale PETN-Behandlung heraufreguliert, die auch zur Heraufregulation der SOD2, GPx1 und eNOS beitragen kann. Im Organbad-Experiment wurde die Acetylcholin-induzierte, Endothel-abhängige Vasodilatation in der Aorta der weiblichen Nachkommen der PETN-Gruppe verstärkt. Diese verbesserte Endothelfunktion, was vermutlich aus der Genexpressionsänderung resultiert, stellt sehr wahrscheinlich einen Schlüsselmechanismus der Blutdrucksenkung in den Nachkommen der PETN-behandelten F0-Tiere dar.

Molekulare Mechanismen Proteinkinase C-induzierter Nox4-Hochregulation in humanen Endothelzellen

Eine verstärkte Transkription von NADPH-Oxidasen (Nox) wird mit der Entstehung von atherosklerotischer Veränderungen in Verbindung gebracht. Die Arbeit unserer Gruppe zeigte, dass die Aktivität der Proteinkinase C (PKC) zu einer Nox4-Hochregulation führt, der dominanten NOX Isoform in endothelialen Zellen. Die vorliegende Arbeit zielte auf die Aufdeckung der dowm-stream gelegenen Mechanismen. Die Behandlung von humanen EA.hy 926-Zellen mit dem PKC Aktivator Phorbol-12-Myristat-13-Acetat (PMA) für 48 h führte in eine signifikante Nox4-mRNA-Hochregulation, welche mittels PKC-Inhibitoren oder PKC alpha siRNA abgewendet werden konnte. PMA führte zu einer andauernden Aktivierung der MAP-Kinase Erk1/2. Die PMA vermittelte Nox4-Expression konnte durch Erk1/2-Inhibitoren oder durch Erk1/2-Knock-down geblockt werden. Down-stream konnte die Involvierung der Erk1/2-Substarte Elk-1 und c-Fos mittels siRNA-Experimente gezeigt werden. Darüber hinaus blockte die Inhibierung der Histondeacetylasen (HDACs) mit Scriptaid oder durch HDAC3-Knock-down mittels siRNA die PMA-induzierte Nox4-Expression in EA.hy 926-Zellen, weswegen eine Rolle für HADC3 in der Regulation der Nox4-Expression angezeigt wurde. Abschließend reduzierte ein Knock-down von p53 (siRNA) deutlich die basale Expression von Nox4, hatte aber nur einen kleinen Effekt auf die PMA-induzierte Nox4-Expression. Zusammenfassend zeigen die Daten der vorliegenden Arbeit, dass in einer PKC alpha induzierten Nox4-mRNA-Hochregulation Erk1/2, Elk-1, cFos und HDAC3 involviert sind.

Rheumatoide Arthritis und Arteriosklerose : Bedeutung der zu Grunde liegenden chronisch entzündlichen Prozesse für die endotheliale Dysfunktion ; Wirkung der antiinflammatorischen Substanz Galiellalacton in murinen Arteriosklerosemodellen

Erhöhte arteriosklerotische und thrombotische Vorfälle sind ein Hauptgrund für die gesteigerten Zahlen kardiovaskulärer Todesfälle von Patienten mit chronisch entzündlichen Erkrankungen wie der rheumatoiden Arthritis (RA). Diese erhöhte Mortalität ist nicht auf die traditionellen Risikofaktoren, wie Alter, Geschlecht, Bluthochdruck oder Diabetes zurückzuführen. Man nimmt an, dass die systemische Entzündung einen nicht-traditionellen Risikofaktor für die erhöhten kardiovaskulären Todesfälle von RA-Patienten darstellt. Da die derzeitige Behandlung der RA zum Teil schwere Nebenwirkungen zur Folge haben kann, war es das Ziel dieser Doktorarbeit, die Zusammenhänge zwischen RA und Arteriosklerose (AS) näher zu untersuchen, sowie die neue antiinflammatorische Substanz Galiellalacton (Gal) für die Behandlung der AS zu charakterisieren.rnIn dem chronisch inflammatorischen Tiermodell der TTP-defizienten Mäuse, dessenrnPhänotyp dem einer humanen RA-Erkrankung ähnelt, konnte eine verschlechterternEndothelfunktion, die als ein erstes Symptom einer erworbenen AS gilt, nachgewiesen werden. Dies konnte auf eine erhöhte Stabilität der Nox2-mRNA zurückgeführt werden, die unabhängig von der erhöhten Expression des Entzündungsmarkers TNFα war. Diese gesteigerte Nox2-Menge führte wiederum zu einer erhöhten Bildung von reaktiven Sauerstoff- und Stickstoffspezies und somit zu einer verringerten Menge an bioaktivem Stickstoffmonoxid, welches die endotheliale Dysfunktion (eDF) bedingte.rnAls ein traditioneller Risikofaktor für das Auftreten von kardiovaskulären Ereignissen gilt unter anderem eine Diabeteserkrankung. Durch die Ausbildung einer Nitrattoleranz bei der Therapie mit organischen Nitraten wie NTG, ISMN oder ISDN kommt es zu der Entwicklung einer eDF. PETN, ein weiteres organisches Nitrat zeigt diese Nebenwirkung nicht. PETN, vermittelt seinen antioxidativen Effekt über die Nrf2-abhängige Induktion der HO-1-Promotoraktivität.rnDie Behandlung von arteriosklerotischen Mäusen (ApoE-/-- und ApoE-/-TFPI+/--Mäuse) mit dem antiinflammatorischen Pilzsekundärmetaboliten Gal zeigte eine verringerte mRNA-Expression von arteriosklerotischen und inflammatorischen Mediatoren, sowie eine reduzierte Thrombenbildung durch eine verringerte Plättchenadhäsion.rnZusammenfassend konnte gezeigt werden, dass inflammationsabhängiger oxidativerrnStress ein Hauptgrund für die entzündungsgetriebene Artheriogenese ist und Galrneine neue Leitsubstanz für die Behandlung dieser Erkrankung ist.

Oxidative damage to DNA related to survivorship and carotenoid-based sexual ornamentation in the common yellowthroat

Carotenoid-based sexual ornaments are hypothesized to be reliable signals of male quality, based on an allocation trade-off between the use of carotenoids as pigments and their use in antioxidant defence against reactive oxygen species. Carotenoids appear to be poor antioxidants in vivo, however, and it is not clear whether variation in ornament expression is correlated with measures of oxidative stress (OXS) under natural conditions. We used single-cell gel electrophoresis to assay oxidative damage to erythrocyte DNA in the common yellowthroat (Geothlypis trichas), a sexually dichromatic warbler in which sexual selection favours components of the males’ yellow ‘bib’. We found that the level of DNA damage sustained by males predicted their overwinter survivorship and was reflected in the quality of their plumage. Males with brighter yellow bibs showed lower levels of DNA damage, both during the year the plumage was sampled (such that yellow brightness signalled current OXS) and during the previous year (such that yellow brightness signalled past OXS). We suggest that carotenoid-based ornaments can convey information about OXS to prospective mates and that further work exploring the proximate mechanism(s) linking OXS to coloration is warranted.

Telomeres: Linking stress and survival, ecology and evolution.

Telomeres are protective structures at the ends of eukaryotic chromosomes. The loss of telomeres through cell division and oxidative stress is related to cellular aging, organismal growth and disease. In this way, telomeres link molecular and cellular mechanisms with organismal processes, and may explain variation in a number of important life-history traits. Here, we discuss how telomere biology relates to the study of physiological ecology and life history evolution. We emphasize current knowledge on how telomeres may relate to growth, survival and lifespan in natural populations. We finish by examining interesting new connections between telomeres and the glucocorticoid stress response. Glucocorticoids are often employed as indices of physiological condition, and there is evidence that the glucocorticoid stress response is adaptive. We suggest that one way that glucocorticoids impact organismal survival is through elevated oxidative stress and telomere loss. Future work needs to establish and explore the link between the glucocorticoid stress response and telomere shortening in natural populations. If a link is found, it provides an explanatory mechanism by which environmental perturbation impacts life history trajectories.

Additive effect of homocysteine- and cholesterol-lowering therapy on endothelium-dependent vasodilation in patients with cardiovascular disease

Endothelial dysfunction is a marker for development and progression of atherosclerosis. Statin therapy improves endothelial function in cardiovascular patients by reducing LDL-cholesterol and by pleiotropic effects. B-group vitamin supplementation restores endothelial function mainly by reducing homocysteine-induced oxidative stress. Thus, we evaluated the effect of rosuvastatin, B-group vitamins and their combination on endothelial function in high-risk cardiovascular patients.

Prenatal Exposure to Testosterone Impairs Oxidative Damage Repair Efficiency in the Domestic Chicken (Gallus Gallus)

Elevated levels of maternal androgens in avian eggs affect numerous traits, including oxidative stress. However, current studies disagree as to whether prenatal androgen exposure enhances or ameliorates oxidative stress. Here, we tested how prenatal testosterone exposure affects oxidative stress in female domestic chickens (Gallus gallus) during the known oxidative challenge of an acute stressor. Prior to incubation, eggs were either injected with an oil vehicle or 5 ng testosterone. At either 17 or 18 days post-hatch, several oxidative stress markers were assessed from blood taken before and after a 20 min acute stressor, as well as following a 25 min recovery from the stressor. We found that, regardless of yolk treatment, during both stress and recovery all individuals were in a state of oxidative stress, with elevated levels of oxidative damage markers accompanied by a reduced total antioxidant capacity. In addition, testosterone-exposed individuals exhibited poorer DNA damage repair efficiencies in comparison with control individuals. Our work suggests that while yolk androgens do not alter oxidative stress directly, they may impair mechanisms of oxidative damage repair.

[Fetal programming of cardiovascular disease: new causes and underlying mechanisms]

There exists an association between pathologic events occurring during early life and the development of cardiovascular disease in adulthood. For example, transient perinatal hypoxemia predisposes to exaggerated hypoxic pulmonary hypertension and preeclampsia predisposes the offspring to pulmonary and systemic endothelial dysfunction later in life. The latter finding offers a scientific basis for observations demonstrating an increased risk for premature cardiovascular morbidity in this population. Very recently, we showed that offspring of assisted reproductive technologies also display generalized vascular dysfunction and early arteriosclerosis. Studies in animal models have provided evidence that oxidative stress and/or epigenetic alterations play an important pathophysiological role in the fetal programming of cardiovascular disease.

Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro

Background Increasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (−NH2) and carboxyl (−COOH) surface modifications. Results Pre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures. Conclusions The present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies. Keywords: Multi-walled carbon nanotubes (MWCNTs); Pulmonary surfactant (Curosurf); Macrophages; Epithelial cells; Dendritic cells; Triple cell co-culture; Pro-inflammatory and oxidative reactions

Postprandial effects of dark chocolate on portal hypertension in patients with cirrhosis: results of a phase 2, double-blind, randomized controlled trial

In cirrhosis, hepatic endothelial dysfunction as a result of oxidative stress contributes to the postprandial increase in hepatic venous pressure gradient (HVPG).

The sodium-dependent ascorbic acid transporter family SLC23

Transporters for vitamin C and its oxidized form dehydroascorbic acid (DHA) are crucial to maintain physiological concentrations of this important vitamin that is used in a variety of biochemical processes. The human SLC23 family consists of the Na(+)-dependent vitamin C transporters SVCT1 (encoded by the SLC23A1 gene) and SVCT2 (SLC23A2) as well as an orphan transporter SVCT3 (SLC23A3). Phylogenetically, the SLC23 family belongs to the nucleobase-ascorbate transporter (NAT) family, although no nucleobase transport has yet been demonstrated for the human members of this family. The SVCT1 and SVCT2 transporters are rather specific for ascorbic acid, which is an important antioxidant and plays a crucial role in a many metal-containing enzymes. SVCT1 is expressed predominantly in epithelial tissues such as intestine where it contributes to the supply and maintenance of whole-body ascorbic acid levels. In contrast to various other mammals, humans are not capable of synthesizing ascorbic acid from glucose and therefore the uptake of ascorbic acid from the diet via SVCT1 is essential for maintaining appropriate concentrations of vitamin C in the human body. The expression of SVCT2 is relatively widespread, where it serves to either deliver ascorbic acid to tissues with high demand of the vitamin for enzymatic reactions or to protect metabolically highly active cells or specialized tissues from oxidative stress. The murine Slc23a3 gene encoding the orphan transporter SVCT3 was originally cloned from mouse yolk sac, and subsequent studies showed that it is expressed in the kidney. However, the function of SVCT3 has not been reported and it remains speculative as to whether SVCT3 is a nucleobase transporter.

Pulmonary hypertension in high-altitude dwellers: novel mechanisms, unsuspected predisposing factors

Studies of high-altitude populations, and in particular of maladapted subgroups, may provide important insight into underlying mechanisms involved in the pathogenesis of hypoxemia-related disease states in general. Over the past decade, studies involving short-term hypoxic exposure have greatly advanced our knowledge regarding underlying mechanisms and predisposing events of hypoxic pulmonary hypertension. Studies in high altitude pulmonary edema (HAPE)-prone subjects, a condition characterized by exaggerated hypoxic pulmonary hypertension, have provided evidence for the central role of pulmonary vascular endothelial and respiratory epithelial nitric oxide (NO) for pulmonary artery pressure homeostasis. More recently, it has been shown that pathological events during the perinatal period (possibly by impairing pulmonary NO synthesis), predispose to exaggerated hypoxic pulmonary hypertension later in life. In an attempt to translate some of this new knowledge to the understanding of underlying mechanisms and predisposing events of chronic hypoxic pulmonary hypertension, we have recently initiated a series of studies among high-risk subpopulations (experiments of nature) of high-altitude dwellers. These studies have allowed to identify novel risk factors and underlying mechanisms that may predispose to sustained hypoxic pulmonary hypertension. The aim of this article is to briefly review this new data, and demonstrate that insufficient NO synthesis/bioavailability, possibly related in part to augmented oxidative stress, may represent an important underlying mechanism predisposing to pulmonary hypertension in high-altitude dwellers.

Expression of HB-EGF by retinal pigment epithelial cells in vitreoretinal proliferative disease

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.

Dual in vitro perfusion of an isolated cotyledon as a model to study the implication of changes in the third trimester placenta on preeclampsia

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-alpha, IL-1ss, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2alpha 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group. Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group. Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1beta of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1beta at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue. Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1beta only the 17kD band was detected. In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

Revascularisation of coronary artery disease in patients with diabetes mellitus

Diabetes mellitus is becoming increasingly prevalent and magnifies the risk of cardiovascular complications. Endothelial dysfunction caused by oxidative stress is a hallmark of diabetes and is responsible for the ubiquitous manifestations of vascular disease in diabetics. Compared with non-diabetic patients, coronary artery disease is more severe and the clinical outcome impaired in diabetic patients undergoing revascularisation. Despite these limitations the benefit of revascularisation therapy is particularly pronounced in diabetics. The optimal revascularisation strategy (coronary artery bypass graft surgery versus percutaneous coronary intervention) in diabetic patients with coronary artery disease depends on clinical and anatomical considerations.