Bernhard Schieffer

Role of NAD(P)H Oxidase in Angiotensin II–Induced JAK/STAT Signaling and Cytokine Induction

Abstract— Inflammatory processes involve both synthesis of inflammatory cytokines, such as interleukin-6 (IL-6), and the activation of their distinct signaling pathways, eg, the janus kinases (JAKs) and signal transducers and activators of transcription (STAT). Superoxide (O2−) anions activate this signaling cascade, and the vasoconstrictor angiotensin II (Ang II) enhances the formation of O2− anions via the NAD(P)H oxidase system in rat aortic smooth muscle cells. Ang II activates the JAK/STAT cascade via its type 1 (AT1) receptor and induces synthesis and release of IL-6. Therefore, we investigated the role of O2− anions generated by the NAD(P)H oxidase system on the Ang II activation of the JAK/STAT cascade and its impact on IL-6 synthesis. Ang II stimulation of rat aortic smooth muscle cells induced a rapid increase in O2− anions determined by laser fluoroscopy, which can be abolished by DPI, a flavoprotein inhibitor. Ang II–induced phosphorylation of JAK2, STAT1&agr;/&bgr;, STAT3, and IL-6-synthesis can be abolished by DPI, as determined by immunoprecipitations and Northern blot analysis. Electroporation of neutralizing antisera targeted against p47phox, a NAD(P)H oxidase subunit, abolished Ang II–induced JAK/STAT activation and IL-6 synthesis. Inhibition of JAK2 by its inhibitor AG490 (10 &mgr;mol/L) blocked not only JAK2 activation but also IL-6 synthesis. These results suggest that stimulation of the JAK/STAT cascade by Ang II requires O2− anions generated by the NAD(P)H oxidase system, and O2− anion–dependent activation of the JAK/STAT cascade seems to be additionally involved in Ang II–induced IL-6 synthesis. Thus, Ang II–induced inflammatory effects seem to require O2− anions generated by the NAD(P)H oxidase system.

Expression of CYR61, an Angiogenic Immediate Early Gene, in Arteriosclerosis and Its Regulation by Angiotensin II

Background—The renin-angiotensin system is thought to be involved in development and progression of arteriosclerosis, thereby contributing to adverse cardiovascular events. To elucidate the role of angiotensin II (Ang II) at a cellular level, we analyzed the Ang II–induced gene expression profile. Methods and Results—Genes induced on Ang II stimulation (10−7 mol/L, 45 minutes) in rat smooth muscle cells were analyzed by polymerase chain reaction selected subtraction. In addition to known genes, such as interleukin 6, leukemia inhibitory factor, and c-fos, we identified CYR61, an angiogenic immediate early gene. Northern blot analysis revealed a rapid 2.5-fold increase of CYR61 transcript levels by Ang II, peaking at 30 minutes, which was blunted by Ang II type 1 receptor blockade. Exposure of rat aortic rings to Ang II (30 minutes) revealed a 2-fold, and intraperitoneal injection of Ang II (30 minutes) in mice a 3-fold, increase of aortic CYR61 transcripts. In arteriosclerotic aortas of apolipoprotein E–deficient mice, CYR61 transcripts confirmed by in situ hybridization and proteins shown by immunohistochemistry were elevated, whereas they were hardly detectable in wild types. In human carotid atherectomies and arteriosclerotic coronary arteries, immunohistochemical analysis revealed expression of CYR61 within connective tissue in neointima, adventitia, and surrounding small capillaries and blood vessels, colocalized with ACE and Ang II. Normal human arteries showed no significant staining for CYR61. Conclusions—CYR61, an angiogenic factor, is induced by Ang II in vascular cells and tissue. The expression of CYR61, colocalized with Ang II and ACE, in small vessels of human arteriosclerotic lesions is consistent with the notion that the activated renin-angiotensin system may contribute to plaque neovascularization by enhancing regulators of microvessel formation and cell proliferation.

5-Lipoxygenase is involved in the angiotensin II-induced NAD(P)H-oxidase activation.

Angiotensin II (ANG II)-induced interleukin (IL)-6 synthesis requires NAD(P)H-oxidase-derived superoxide anions. Since NAD(P)H-oxidase activation by cytokines involves 5-lipoxygenase (LOX)-derived leukotriene B(4) (LTB(4)) formation, we postulated that LTB(4) is involved in the ANG II-dependent NAD(P)H-oxidase activation. Therefore, 5-LOX expression and LTB(4) formation following ANG II (100nM) stimulation were determined in rat aortic smooth muscle cells (SMC). Reactive oxygen species (ROS)-formation and IL-6 mRNA expression were analyzed following ANG II and LTB(4) (0.6 microM) stimulation. 5-LOX mRNA and protein were detected in SMC. ANG II-induced LTB(4) formation at 2.5min and was followed by an increase in ROS-formation and IL-6 mRNA expression. Blockade of 5-LOX by MK886 (200nM) abrogated LTB(4)-formation, ROS-formation, and IL-6 mRNA expression. Moreover, LTB(4)-induced ROS-formation and IL-6 mRNA expression was abolished by NAD(P)H-oxidase inhibition using diphenyleneiodonium chloride (DPI 10 microM). In conclusion, the present study demonstrates that ANG II enhances LTB(4)-formation in an 5-LOX dependent manner. LTB(4) activates the vascular type NAD(P)H-oxidase, leading to an increase in IL-6 transcripts.

Role of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors, angiotensin-converting enzyme inhibitors, cyclooxygenase-2 inhibitors, and aspirin in anti-inflammatory and immunomodulatory treatment of cardiovascular diseases.

The immunologic response in atherosclerosis involves not only intrinsic cells of the artery wall, but also circulating leukocytes, lymphocytes, and macrophages. Interaction of various arms of the immune response modulates plaque development and stability, and it is conceivable that immunologic effects of some cardiovascular therapies may contribute to their mechanism of benefit. The preponderance of data has accrued with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins). Statin effects, such as inhibition of T cell activation, tissue factor expression, or reduction of platelet hyperreactivity, may elicit beneficial effects in vitro and in vivo in patients with coronary artery disease. Moreover, aspirin may limit oxidation of lipoproteins and fibrinogen, and it may inhibit cytokine-induced nitric oxide synthase II expression. The hypothesis that selective inhibition of cyclooxygenase-2 (COX-2) may increase risk of myocardial infarction is controversial and may also be of questionable clinical significance. Finally, angiotensin-converting enzyme (ACE) inhibitors not only reduce proinflammatory mediators, such as interleukin-6, but also enhance the concentration of anti-inflammatory cytokines, such as interleukin-10. Because ACE is expressed at the shoulder region of atherosclerotic plaques, and ACE activity is enhanced in unstable plaques, ACE inhibition may also contribute to plaque stability. This article reviews the potential immunomodulatory potencies of aspirin, COX-2 inhibitors, statins, and ACE inhibitors as established pharmacotherapy in patients with coronary artery disease.

Transcatheter Closure of Patent Foramen Ovale in Patients with Paradoxical Embolism. Procedural and Follow‐up Results after Implantation of the Starflex® Occluder Device with Conjunctive Intensified Anticoagulation Regimen

BACKGROUNDnPrevalence of patent foramen ovale (PFO) is higher in patients with paradoxical embolism and associated with increased risk for recurrent thromboembolic events. By percutaneous closure of PFO, surgical closure or permanent oral anticoagulation can be avoided. So far, published series included different occluder systems and various indications and regimens of postprocedural anticoagulation. The aim of the present study was to evaluate the short- and long-term results after implantation of the Starflex occluder in patients with PFO using an intensified anticoagulation regimen.nnnMETHODS AND RESULTSn154 patients with PFO (94 men; age: 44 +/- 13 years) and >or=1 thromboembolic event were included. Other causes for embolism were excluded. PFO closure was successful in 147 patients (95.5%). All patients were treated with phenprocoumon (INR 2.5) and aspirin (100 mg/die) for 6 months. Transesophageal echocardiography (TEE) was repeated at 6 months. Mean clinical follow-up period was 26 +/- 18 months. After 6 months, five patients had a significant residual shunt, and five patients had suspected thrombus formation on the occluder. In three of these five patients, the occluder was surgically removed and foreign body reaction was noted. During follow-up, nine patients suffered from neurological events (two strokes, seven transient ischemic attacks [TIA]), though complete closure of the PFO was documented by TEE. Two patients died during follow-up; three patients had bleeding complications.nnnCONCLUSIONnPercutaneous closure of PFO in symptomatic patients by Starflex occluder represents an effective therapy with a low incidence of periinterventional complications and recurrent thromboembolism. However, thrombus formation at the occluder system may occur in some patients despite an aggressive anticoagulation regimen.

ACE Gene Polymorphism and Coronary Artery Disease : A Question of Persuasion or Statistical Confusion?

“In the intermediate phase, swiftly developing complexity within the system hides the risk of imminent chaos. But the risk is there.” Michael Chrichton’s The Lost World nnGreat personal enthusiasm, temptation, technical efforts, and complex mathematical equations characterize the developing research field of newly identified gene polymorphisms and their impact on a variety of cardiovascular diseases. After the first publication of Cambien and coworkers1 in Nature in 1992, who reported that the D / D angiotensin-converting enzyme (ACE) polymorphism is a potent risk factor for myocardial infarction, great enthusiasm emerged that a potentially new class of risk factors was identified. In the following years, researchers repeatedly tried to prove with controversial results that a single-gene polymorphism, such as the ACE polymorphism, is associated with a higher risk of myocardial infarction, for example. Much to their surprise, different populations apparently responded differently with regard to the appearance and overall impact of the ACE polymorphism on myocardial infarction. One of the potential answers is related to the composition of polymorphisms in different study populations. Whereas Cambien et al analyzed a population in which ≈50% were ACE D / D carriers, the distribution of this polymorphism was found to be only ≈23% in normal Western populations.nnIn this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Agerholm-Larsen and coworkers2 report results from a meta-analysis of the ACE gene polymorphism studies …

Impact of a combined treatment of angiotensin II type 1 receptor blockade and 3-hydroxy-3-methyl-glutaryl-CoA-reductase inhibition on secretory phospholipase A2-type IIA and low density lipoprotein oxidation in patients with coronary artery disease

AIMSnTo evaluate the impact of a combined treatment of angiotensin II type 1 (AT(1))-receptor blockade and 3-hydroxy-3-methyl-glutaryl-CoA-reductase inhibition (statin) on the secretory phospholipase A(2) type IIA (sPLA(2)-IIA) and oxidized low density lipoprotein (oxLDL) in patients with coronary artery disease (CAD).nnnMETHODS AND RESULTSnSixty patients with angiographically documented CAD and a history of arterial hypertension were randomized in a double-blinded fashion to pravastatin (PRAV, 40 mg/day, n = 30) or PRAV plus irbesartan (PRAV+IRB, 40 mg/day+300 mg/day, n = 30) and were treated for 3 months. Blood pressure (BP) and cholesterol fractions were determined at baseline and after 3 months. SPLA(2) activity as primary endpoint, sPLA(2)-IIA protein, oxLDL levels, and high-sensitivity (hs)-C-reactive protein were measured by an enzyme-linked immunabsorbent assay. In both treatment groups, systolic BP levels and circulating HDL and LDL levels were reduced to the same extent. The combined treatment of PRAV+IRB significantly decreased sPLA(2)-IIA activity and sPLA(2)-IIA-protein concentration compared with PRAV treatment alone (P < 0.05). In addition, PRAV+IRB significantly reduced oxLDL levels compared with PRAV treatment alone (P < 0.05). This effect was independent of changes in LDL cholesterol levels.nnnCONCLUSIONnThese findings are consistent with the notion that the combined treatment of pravastatin with irbesartan reduced sPLA(2)-IIA-activity, sPLA(2)-IIA-protein concentration, and oxLDL in patients with CAD suggesting a novel anti-atherogenic effect by combining AT(1)-receptor blockade with statin treatment.

TNFα decreases αMHC expression by a NO mediated pathway: role of E-box transcription factors for cardiomyocyte specific gene regulation

Objective: Tumor necrosis factor a (TNFa) is thought to play a key role in the pathogenesis of cardiac failure. In the myocardium, TNFa enhances the expression of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) has been shown to affect b-agonistdependent cardiac contractility and relaxation. It is not clear, however, whether TNF a mediated NO release has sustained cardiac effects, by altering expression of cardiomyocyte specific genes such as a-myosin heavy chain (aMHC). Methods: Neonatal rat ventricular cardiomyocytes (CM) were stimulated with TNFa and / or the NOS inhibitor nitro-L-arginine (L-NNA). Protein binding to the E-box enhancer element in the aMHC promoter was evaluated by electrophoretic mobility shift assay (EMSA) and transcriptional activity of the E-box consensus motif was determined by luciferase assay. mRNA levels of the endogenous aMHC gene were assessed by RT‐PCR. In vivo studies were performed in transgenic mice with cardiac specific over-expression of TNFa. Results: CM treated with TNFa exhibited decreased levels of aMHC transcripts (6968% of control), the effect of TNFa was reversed by L-NNA (94614% of control). As shown by EMSA, TNFa reduced protein binding to the aMHC E-box enhancer motif via NO dependent pathways. Addition of the NO-donor sodium nitroprusside (SNP) to CM nuclear extracts dose dependently disrupted protein binding to the aMHC E-box. Furthermore, exposure of CM to TNFa or SNP decreased transcription from an E-box luciferase-reporter construct (TNFa :7 4 612%; SNP 250 mM: 72610%; SNP 500 mM: 66611% of control). In myocardial tissue of TNFa transgenic mice, increased nitrotyrosine staining, decreased protein binding to the aMHC E-box motif and reduced expression of aMHC (62626%) were observed. Conclusions: The present study shows that TNFa reduces aMHC transcript levels in cardiomyocytes. Our data obtained in cultured CM and in TNFa transgenic mice support the notion that TNFa exerts these effects by NO and E-box dependent mechanisms in vitro and possibly in vivo.

Transcatheter Closure of Patent Foramen Ovale (PFO) in Patients with Paradoxical Embolism: Procedural and Follow-Up Results after Implantation of the Amplatzer®-Occluder Device

BACKGROUNDnPrevalence of patent foramen ovale (PFO) with detectable right-to-left shunt is higher in young adults with transient ischemic attack (TIA) and stroke compared to the general population. So far, published series included different occluder systems, various indications and regimens of postprocedural anticoagulation. In our experience, occluder systems may be associated with an increased prevalence of thrombus formation, which has also reported by other groups. The aim of the present study was to evaluate the follow-up results after implantation of the Amplatzer® occluder in patients with PFO using a consistent anticoagulation regimen.nnnMETHODS AND RESULTSnOne-hundred and fourteen patients with PFO (60 men; age: 47 ± 13 years) and ≥1 thromboembolic event were included. Other causes for embolism were excluded. PFO-closure was successful in all patients. All patients were treated with aspirin (100 mg/day) and clopidogrel (75 mg/day) for 6 months. TEE was repeated at a mean of 10.3 months. Mean clinical follow-up period was 18 ± 9 months. After a mean of 10 months, no patient had either a significant residual shunt nor a suspected thrombus formation on the occluder. During follow-up, 5 patients suffered from neurological events (1 stroke, 2 TIAs, 2 epileptic seizures), though complete closure of the PFO was documented by TEE. One patient suffered from bleeding complications (upper GI-bleeding).nnnCONCLUSIONnPercutaneous closure of PFO in symptomatic patients by Amplatzer® occluder represents an effective therapy with a low incidence of peri-interventional complications and recurrent thromboembolism. Thrombus formations on the occluder system were not detected in this cohort.

Albuminuria: pathophysiology, epidemiology and clinical relevance of an emerging marker for cardiovascular disease

Albuminuria has emerged from being a sign of early kidney disease in diabetes to an independent predictor of cardiovascular risk. Epidemiological studies suggest that microalbuminuria (30-300 mg/d) is present in 5-19% of the general population, in up to 23% of patients with hypertension and in up to 40% of patients with diabetes. Recent data suggest an even higher prevalence in certain patient populations. As it is associated with a variety of important cardiovascular risk factors, including prediabetes, dyslipidemia and the metabolic syndrome, detection of albumin in the urine represents an important diagnostic window for systemic micro- or macrovascular damage. Various studies have demonstrated the predictive value of all levels of albuminuria for future cardiovascular events in patients with diabetes, hypertension or overt cardiovascular disease, as well as in the general population. Annual screening for microalbuminuria is now recommended by international diabetes guidelines for patients with diabetes, and may be appropriate for nondiabetics with risk factors for cardiovascular disease.