Cornelius Müller

Redox-sensitive vascular smooth muscle cell proliferation is mediated by GKLF and Id3 in vitro and in vivo

Reactive oxygen species such as super‐oxide and hydroxyl radicals have been implicated in the pathogenic growth of various cell types. The molecular mechanisms involved in redox‐sensitive cell growth control are poorly understood. Stimulation of cultured vascular smooth muscle cells (VSMC) with xanthin/ xanthin oxidase (X/XO) increases proliferation, whereas stimulation with hydrogen peroxide and Fe3+NTA (H‐Fe) causes growth arrest of VSMC. Differential Display led to the identification of two novel, differentially regulated redox‐sensitive genes. The dominant negative helix‐loop‐helix protein Id3 is induced by X/XO and down‐regulated by H‐Fe. The transcription factor gut‐enriched Kruppel‐like factor (GKLF) is induced by H‐Fe but not by X/XO. Induction of GKLF and inhibition of Id3 via transfection experiments leads to growth arrest, whereas overexpression of Id3 and inhibition of GKLF cause cell growth. Id3 down‐regulation is induced via binding of GKLF to the Id3 promotor and concomitantly reduced Id3 gene transcription rate. GKLF induction by H‐Fe is mediated through hydroxyl radicals, p38MAP kinase‐, calcium‐, and protein synthesis‐dependent pathways. Id3 is induced by X/XO via superoxide, calcium, p38, and p42/44 MAP kinase. GKLF induces and Id3 depresses expression of p21WAF1/Cip1, p27KIP1, p53. Induction of Id3 is accomplished by angiotensin II via superoxide release. A vascular injury mouse model revealed that Id3 is overexpressed in proliferating vascular tissue in vivo. These findings reveal novel mechanisms of redoxcontrolled cellular proliferation involving GKLF and Id3 that may have general implications for our understanding of vascular and nonvascular growth control.—Nickenig, G., Baudler, S., Müller, C., Werner, N., Welzel, H., Strehlow, K., Böhm, M. redox‐sensitive vascular smooth muscle cell proliferation is mediated by GKLF and Id3 in vitro and in vivo. FASEB J. 16, 1077–1086 (2002)

Effects of a quercetin-rich onion skin extract on 24 h ambulatory blood pressure and endothelial function in overweight-to-obese patients with (pre-)hypertension: a randomised double-blinded placebo-controlled cross-over trial

The polyphenol quercetin may prevent CVD due to its antihypertensive and vasorelaxant properties. We investigated the effects of quercetin after regular intake on blood pressure (BP) in overweight-to-obese patients with pre-hypertension and stage I hypertension. In addition, the potential mechanisms responsible for the hypothesised effect of quercetin on BP were explored. Subjects (n 70) were randomised to receive 162 mg/d quercetin from onion skin extract powder or placebo in a double-blinded, placebo-controlled cross-over trial with 6-week treatment periods separated by a 6-week washout period. Before and after the intervention, ambulatory blood pressure (ABP) and office BP were measured; urine and blood samples were collected; and endothelial function was measured by EndoPAT technology. In the total group, quercetin did not significantly affect 24 h ABP parameters and office BP. In the subgroup of hypertensives, quercetin decreased 24 h systolic BP by −3·6 mmHg (P=0·022) when compared with placebo (mean treatment difference, −3·9 mmHg; P=0·049). In addition, quercetin significantly decreased day-time and night-time systolic BP in hypertensives, but without a significant effect in inter-group comparison. In the total group and also in the subgroup of hypertensives, vasoactive biomarkers including endothelin-1, soluble endothelial-derived adhesion molecules, asymmetric dimethylarginine, angiotensin-converting enzyme activity, endothelial function, parameters of oxidation, inflammation, lipid and glucose metabolism were not affected by quercetin. In conclusion, supplementation with 162 mg/d quercetin from onion skin extract lowers ABP in patients with hypertension, suggesting a cardioprotective effect of quercetin. The mechanisms responsible for the BP-lowering effect remain unclear.

Post-transcriptional regulation of the AT1 receptor mRNA. Identification of the mRNA binding motif and functional characterization

The expression of the angiotensin AT1 receptor is regulated predominately via post‐transcriptional mechanisms. Angiotensin II reduces the AT1 receptor mRNA half‐life from approximately 6 to 2 h, as assessed by experiments under transcriptional blockade and by in vitro mRNA decay assays in vascular smooth muscle cells (VSMC). UV light‐treated mRNA‐protein cross‐link assays revealed that the AT1 receptor mRNA interacts with at least six polysomal proteins at its 3′‐end. Multiple mutational studies demonstrated that several binding proteins associated with the AT1 receptor mRNA at bases 2175‐2195. Stimulation of VSMC with angiotensin II significantly enhanced the binding of 50 and 60 kDa proteins to the 3′‐untranslated region of the AT1 receptor mRNA. Transfection decoy assays with AT1 receptor mRNA transcript bases 1864‐2213, 2175‐2213, and 2175‐2195 led to a significantly increased basal expression of AT1 receptor mRNA and inhibited angiotensin II‐induced AT1 receptor mRNA down‐regulation. In vitro decay assays revealed that competition of binding of polysomal proteins to bases 2175‐2195 stabilizes the AT1 receptor mRNA. Polysomal proteins thus bind to the 3′‐untranslated region of the AT1 receptor mRNA at the nucleotide sequence 2175‐2195. This protein‐mRNA interaction is decisively involved in the destabilization of the AT1 receptor mRNA by angiotensin II.

Insulin-like growth factor induces up-regulation of AT1-receptor gene expression in vascular smooth muscle cells

Background Insulin-like growth factor-1 (IGF-1), as well as AT1-receptor activation, plays a central role in growth processes of cardiac and vascular cells. In order to assess relevant interactions of both systems, the effect of IGF-1 on AT1-receptor expression was evaluated in vascular smooth muscle cells. Methods and results Incubation of cultured vascular smooth muscle cells (VSMC) with IGF-1 led to a dose- and time-dependent up-regulation of AT1-receptor mRNA, as measured by Northern hybridisations. The maximal AT1-receptor overexpression of 201±70% of control levels was reached after a 24-hour incubation with 100 ng/ml IGF-1. Consequently, AT1-receptor protein expression was increased to 231±35% of control levels. Experiments under transcriptional blockade showed that AT1-receptor mRNA stability was not altered by IGF-1, suggesting that transcriptional mechanisms may be involved in IGF-1-induced AT1-receptor regulation. Preincubation with various pharmacological inhibitors revealed that IGF-1 up-regulated AT1-receptor expression via activation of p42/44 MAP kinase, whereas tyrosine phosphorylation and PI-3 kinase seemed not to participate in this regulative pathway. Conclusions IGF-1-induced up-regulation of the AT1-receptor may be an important interaction by which cellular growth is modulated in the heart as well as in the vasculature. This may have implications for the treatment regimen of patients suffering from hypertension, cardiac hypertrophy, and coronary heart disease.

Doppler-based renal resistance index for the detection of acute kidney injury and the non-invasive evaluation of paravalvular aortic regurgitation after transcatheter aortic valve implantation

AIMS Acute kidney injury (AKI) is a strong but rather late predictor of mortality after transcatheter aortic valve implantation (TAVI). Early clinically useful markers for the detection of AKI and prediction of outcome are needed in order to control and improve management of periprocedural complications after TAVI. The aim of our study was to assess the predictive value of the Doppler-based renal resistance index (RRI), which correlates inversely with effective renal blood flow and creatinine clearance, for AKI in patients undergoing TAVI and to evaluate its association with paravalvular aortic regurgitation (PAR). METHODS AND RESULTS TAVI was performed with the Medtronic CoreValve prosthesis in 132 consecutive high-risk patients (mean logistic EuroSCORE: 30.3±18.2%). RRI, serum creatinine and cystatin C level were determined before, and 4 hrs, 24 hrs, 48 hrs, 72 hrs, and 7 days after TAVI. AKI occurred in 32/132 patients (24.2%). While serum creatinine and cystatin C levels decreased at first after TAVI (also in most patients developing AKI), the RRI increased significantly immediately after the procedure from 0.79±0.09 to 0.87±0.12 in patients developing AKI (p=0.003). A RRI >0.85 predicted post-interventional AKI with a sensitivity of 58% and specificity of 86%, and was superior to the serum creatinine level (p<0.001). In addition, an elevated RRI was significantly related to haemodynamic changes after TAVI and was associated with the occurrence of moderate/severe PAR (p<0.001). CONCLUSIONS Measurement of the Doppler-based RRI predicts risk for AKI and increased mortality rates at an early post-procedural time point and is related to the occurrence of more-than-mild paravalvular aortic regurgitation after TAVI.

Estrogen improves vascular function via peroxisome-proliferator-activated-receptor-γ

The exact mechanism of estrogen in cardiovascular disease is not fully understood. As estrogen receptors (ERs), the peroxisome-proliferator-activated-receptor-γ (PPARγ) belongs to the family of ligand activated nuclear receptors regulating atheroprotective genes. The aim of this project was to investigate whether vascular effects of estrogen are mediated via PPARγ-regulation in the vascular compartment. Estrogen deficient ovariectomized wildtype-mice (OVX) displayed significant reduction of PPARγ-expression in aortic tissue compared to wildtype-mice with intact ovarian function (Sham). Hormone replacement with subdermal 17ß-estradiol pellets significantly increased vascular PPARγ-expression in ovariectomized female wildtype-mice (OVX/E2). Analogous to wildtype-mice, estrogen-deficient OVX ApoE(-/-)-mice had low vascular PPARγ-expression associated with ROS generation, endothelial dysfunction and atherogenesis. Estrogen replacement (OVX/E2) rescued vascular PPARγ-expression, reduced ROS generation, monocyte recruitment, atherosclerotic lesion formation and improved endothelial function. Inhibition of PPARγ by GW9662, a specific PPARγ-antagonist reduced 17ß-estradiol mediated vascular effects (OVX/E2+GW9662). Finally, despite estrogen deficiency treatment with pioglitazone (OVX+pioglitazone), a selective PPARγ-agonist, compensates deterioration of vascular morphology and function. 17ß-estradiol regulates vascular PPARγ-expression in wildtype- and ApoE(-/-)-mice. The presented data demonstrate the fundamental relevance of PPARγ as downstream target of 17ß-estradiol-related anti-inflammatory and atheroprotective effects within the vascular wall independent of its cardiovascular risk factor modifications.

The prognostic value of acute and chronic troponin elevation after transcatheter aortic valve implantation

AIMS Myocardial injury occurs frequently following transcatheter aortic valve implantation (TAVI). The aim of this study was to assess timing, predictors, and prognostic value of periprocedural myocardial injury and chronic troponin elevation after TAVI. METHODS AND RESULTS Two hundred and seventy-six patients (logistic EuroSCORE 26.6±17.1%) underwent transvascular TAVI. Troponin, CK-MB, and NT-proBNP levels were measured before and after TAVI (1 hr, 4 hrs, 24 hrs, 48 hrs, 72 hrs, seven days, three, and six months). Myocardial injury (according to VARC-2 recommendation defined as ΔTroponin ≥15x URL) occurred in 143/276 patients (51.8%) during the first 72 hours following TAVI. Use of a self-expanding prosthesis (p=0.02), coronary artery disease (p=0.04), higher left ventricular ejection fraction (LVEF) (p<0.001), and procedure time (p<0.001) were independent predictors for the development of myocardial injury after TAVI. Thirty-day (4.2% vs. 6.1%; p=0.48) and one-year mortality (19.4% vs. 26.5%; p=0.15) were not related to the incidence of periprocedural myocardial injury. However, patients with chronic troponin elevation after TAVI had an increased one-year mortality risk (HR 4.5, 95% CI: 2.0-10.0; p<0.001). CONCLUSIONS Myocardial injury defined as ΔTroponin ≥15x URL after TAVI seems to be a procedure-related issue without impact on 30-day and one-year survival. However, monitoring of post-procedural troponin might be useful for prognostication after TAVI.

Circulating Microparticles Decrease After Cardiac Stress in Patients With Significant Coronary Artery Stenosis.

Cardiac stress leads to a dynamic increase of circulating microparticles (MPs) in healthy individuals that is diminished in individuals with vascular disease. The impact of coronary ischemia on circulating MP level is unknown. This study investigates the kinetics of circulating MPs during cardiac stress in patients with coronary artery stenosis.

Genetic disruption of multidrug resistance-associated protein 1 improves endothelial function and attenuates atherosclerosis in MRP1 –/– LDLr –/– double knockout mice

Introduction Multidrug resistance-associated protein 1 (MRP1) is an anion transporter which is implicated in the efflux of the intracellular antioxidant anion glutathione as well as leukotrienes. Pharmacological inhibition of MRP1 exhibits antioxidative and anti-atherosclerotic effects both in vitro and in vivo. However, pharmacological inhibitors of MRP1 lack selectivity, which prompted us to study the in vivo impact of a genetic disruption of MRP1 on endothelial dysfunction, reactive oxygen species formation and atherogenesis in an atherosclerotic mouse model. Material and methods MRP1−/− LDLr−/− double knockout mice. were fed a high-fat and cholesterol-rich diet for 7 weeks. Thereafter, endothelial function was assessed in isolated aortic rings. Reactive oxygen species were quantified by L-012 chemiluminescence, and the atherosclerotic plaque burden was measured following oil red O staining. Results Endothelium-dependent vasodilation of MRP1−/− LDLr−/− double knockout mice was significantly improved compared to MRP1-competent LDLr−/− single knockout mice (0.56 ±0.06 vs. 0.78 ±0.08; n = 10; p = 0.048). This improvement was accompanied by a significant reduction in reactive oxygen species formation within the aortic tissue (102 ±27 RLU/s/mg vs. 315 ±78 RLU/s/mg, n = 9–11, p = 0.03). Moreover, the atherosclerotic plaque burden of MRP1−/− LDLr−/− double knockout mice was significantly reduced (0.06 ±0.01 vs. 0.12 ±0.02; n = 6; p = 0.047). Finally, arterial blood pressure was significantly reduced in MRP1−/− LDLr−/− double knockout mice (93 ±5 mm Hg vs. 128 ±4 mm Hg; n = 8–12; p < 0.001). Conclusions Genetic disruption of MRP1 appears to reduce blood pressure and vascular oxidative stress in vivo, which leads to improved endothelial function and a reduced plaque burden in atherosclerotic mice. Therefore, MRP1 might represent a promising therapeutic target to improve endothelial function in patients suffering from atherosclerosis.