Martin Spiecker

Inhibition of Endothelial Vascular Cell Adhesion Molecule-1 Expression by Nitric Oxide Involves the Induction and Nuclear Translocation of IκBα

The induction of vascular cell adhesion molecule-1 (VCAM-1) expression by tumor necrosis factor (TNF)-α requires the activation of nuclear factor-κB (NF-κB) via a process involving the phosphorylation and degradation of its cytoplasmic inhibitor, IκBα. We have shown that nitric oxide (NO) decreases VCAM-1 expression via inhibition of NF-κB activation. To determine how NO inhibits NF-κB, we studied the fate of IκBα following TNF-α stimulation in the presence of NO donorsS-nitrosoglutathione and sodium nitroprusside. Activation of NF-κB by TNF-α occurred within 15 min and coincided with rapid degradation of IκBα. Co-treatment with NO donors did not prevent IκBα phosphorylation or degradation. However, after 2 h of TNF-α stimulation, NO donors inhibited NF-κB activation and augmented IκBα resynthesis and nuclear translocation by 2.5- and 3-fold, respectively. This correlated with a 75% reduction in TNF-α-induced VCAM-1 expression. In a time-dependent manner, NO donors alone caused the nuclear translocation of IκBα. To confirm that NO donors have similar effects as endogenously derived NO, murine macrophage-like cells, RAW264.7, were co-cultured with endothelial cells. Induction of RAW264.7-derived NO inhibited lipopolysaccharide-induced endothelial VCAM-1 expression, which was reversed by the NO synthase inhibitorN ω-monomethyl-l-arginine. These findings indicate that NO inhibits NF-κB activation and VCAM-1 expression by increasing the expression and nuclear translocation of IκBα.

Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants.

Although nitric oxide (NO) and antioxidants inhibit adhesion molecule expression, their inhibitory effects on nuclear factor κB (NF‐κB) activation may differ. The NO donors, but not 8‐bromo‐cGMP, decreased tumor necrosis factor α (TNF‐α)‐induced VCAM‐1, ICAM‐1, and E‐selectin expression by 11‐70%. In contrast, NAC completely abolished VCAM‐1 and E‐selectin expression and decreased ICAM‐1 expression by 56%. Gel shift assays demonstrate that NF‐κB activation was inhibited by both NO and antioxidants. The activation of NF‐κB involves the phosphorylation and degradation of its cytoplasmic inhibitor IκB‐α by 26S proteasomes. The 26S proteasome inhibitor MG132 prevented the degradation of phosphorylated IκB‐α. NAC inhibited IκB kinase (IKK) activity and prevented IκB‐α phosphorylation and degradation. In contrast, NO did not inhibit IKK activity, IκB‐α phosphorylation, or IκB‐α degradation. However, NO, but not antioxidants, induced IκB‐α promoter activity. The inhibitory effects of NO on adhesion molecule expression, therefore, differs from that of antioxidants in terms of the mechanism by which NF‐κB is inactivated. J. Leukoc. Biol. 63: 732–739; 1998.

Risk of Coronary Artery Disease Associated With Polymorphism of the Cytochrome P450 Epoxygenase CYP2J2

Background—Cytochrome P450 (CYP) 2J2 is expressed in the vascular endothelium and metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs). The EETs are potent endogenous vasodilators and inhibitors of vascular inflammation. However, it is not known whether genetic polymorphisms of CYP2J2 are associated with increased cardiovascular risks. Methods and Results—All 9 exons of the CYP2J2 gene and its proximal promoter were sequenced in 132 patients to identify potential variants. Functional consequence of a single nucleotide polymorphism (SNP) in the promoter of CYP2J2 was further evaluated by use of transcription factor-binding and reporter assays. A total of 17 polymorphisms were identified. One of the most relevant polymorphisms in terms of frequency and functional importance is located at −50 (G-50T) in the proximal promoter of CYP2J2. Screening of 289 patients with coronary artery disease and 255 control subjects revealed 77 individuals with the G-50T SNP (17.3% of coronary artery disease patients, 10.6% of control subjects; P=0.026). The association of the G-50T polymorphism remained significant after adjustment for age, gender, and conventional cardiovascular risk factors (OR, 2.23; 95% CI, 1.04 to 4.79). The G-50T mutation resulted in the loss of binding of the Sp1 transcription factor to the CYP2J2 promoter and resulted in a 48.1±2.4% decrease in CYP2J2 promoter activity (P<0.01). Plasma concentrations of stable EET metabolites were significantly lower in individuals with the G-50T SNP. Conclusions—A functionally relevant polymorphism of the CYP2J2 gene is independently associated with an increased risk of coronary artery disease.

A Functional Role of IκB-ε in Endothelial Cell Activation

The NF-κB inhibitor IκB-ε is a new member of the IκB protein family, but its functional role in regulating NF-κB-mediated induction of adhesion molecule expression is unknown. In vascular endothelial cells, IκB-ε associates predominantly with the NF-κB subunit Rel A and to a lesser extent with c-Rel, whereas IκB-α and IκB-β associate with Rel A only. Following stimulation with TNF-α, pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine, and dexamethasone prevented IκB kinase-induced IκB-α, but not IκB-β or IκB-ε phosphorylation and degradation. Since the activation of NF-κB is required for the induction of adhesion molecule expression, we examined the role of IκB-ε in the transactivation of promoters from VCAM-1, ICAM-1, and E-selectin. Using reporter gene constructs of adhesion molecule promoters, PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 promoter activity. Subcloning of κB cis-acting elements of VCAM-1, E-selectin, and ICAM-1 into a heterologous promoter construct revealed that PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 κB promoter activity. By electrophoretic mobility shift assay, NF-κB heterodimers containing c-Rel specifically bind to the κB motif in the ICAM-1, but not VCAM-1 or E-selectin promoter. Indeed, overexpression of c-Rel induced ICAM-1 κB promoter activity to a greater extent than that of E-selectin and overexpression of IκB-ε inhibited ICAM-1 and VCAM-1 promoter activity in endothelial cells. These findings indicate that c-Rel-associated IκB-ε is involved in the induction of ICAM-1 expression.

NO Reduces PMN Adhesion to Human Vascular Endothelial Cells Due to Downregulation of ICAM-1 mRNA and Surface Expression

Reperfusion damage is largely due to the adherence of polymorphonuclear leukocytes to the endothelium initiated by adhesion molecule upregulation. The reduced endothelial nitric oxide release during ischemia may be involved in the upregulation of intercellular adhesion molecule 1. In this study, we tested if nitric oxide donors suppress polymorphonuclear leukocyte adherence to activated endothelial cells by inhibition of the intercellular adhesion molecule 1 surface expression. Confluent human umbilical vein endothelial cells were stimulated with tumor necrosis factor alpha (300 U/mL) after preincubation with increasing concentrations of the nitric oxide donors CAS 1609 (0.005-5 mM/L) and 3-(4-morpholinyl)-sydnonimine (0.01-1 mM/L). Intercellular adhesion molecule 1 surface expression was measured in a cell surface enzyme-linked immunosorbent assay, intercellular adhesion molecule 1 mRNA by Northern analysis. Human saphenous vein endothelial cells were transfected with the inducible nitric oxide synthase gene and stimulated with tumor necrosis factor alpha (300 U/mL). Fluorescein green-labeled polymorphonuclear leukocytes adhering to activated human umbilical vein endothelial cells/human saphenous vein endothelial cells were quantified by epifluorescent microscopy. The intercellular adhesion molecule 1 surface expression of activated human umbilical vein endothelial cells/human saphenous vein endothelial cells was significantly diminished to 40 to 60% of the maximum after treatment with CAS 1609, 3-(4-morpholinyl)-sydnonimine, or transfection with the inducible nitric oxide synthase gene. Intercellular adhesion molecule 1 mRNA was diminished by CAS 1609 and 3-(4-morpholinyl)-sydnonimine in the same manner. The functional relevance of our data was shown by reduction of polymorphonuclear leukocyte adherence to activated human umbilical vein endothelial cells/human saphenous vein endothelial cells following treatment with CAS 1609 and 3-(4-morpholinyl)-sydnonimine or transfection with inducible nitric oxide synthase. Tumor necrosis factor-induced polymorphonuclear leukocyte adherence was abolished by blocking antibody against intercellular adhesion molecule 1. Thus, exogenous or endogenous substitution of nitric oxide diminishes the expression of endothelial intercellular adhesion molecule 1 and its mRNA following tumor necrosis factor alpha stimulation. This results in a reduced polymorphonuclear leukocyte adherence to activated endothelium.

Quantitative assessment of aortic stenosis by three-dimensional echocardiography☆☆☆★

The purpose of this study was to assess the feasibility of three-dimensional echocardiography in aortic stenosis. Planimetric determination of valve area and dynamic volume-rendered display were performed. Three-dimensional echocardiography permits display of any desired plane of the cardiac structure. Thus in the case of aortic stenosis, the plane used for planimetric evaluation can be positioned exactly through the valve orifice. Dynamic volume-rendered display may provide a spatial demonstration of the stenotic valve. In 48 patients aortic valve area was measured by planimetry. The three-dimensional data set was acquired by a workstation in the course of a multiplane transesophageal examination. Results were compared with those obtained by multiplane transesophageal two-dimensional planimetric technique and invasive measurement. A dynamic three-dimensional reconstruction was displayed. Planimetric determination of valve area was possible in 42 (88%) of 48 cases. Statistical analysis of the data acquired showed a good agreement between three-dimensional echocardiography and transesophageal echocardiography (mean difference +0.018 cm2; SD = 0.086) and between three-dimensional echocardiography and the invasive technique (mean difference +0.012 cm2; SD = 0.12). Dynamic volume-rendered display was possible in 42 of 48 cases. Three-dimensional echocardiography permits accurate and reliable determination of aortic valve area. Preoperative spatial recognition of the stenotic valve is possible by dynamic volume-rendered display.

Comparison of Saruplase and Alteplase in Acute Myocardial Infarction

Four hundred seventy-three patients with acute myocardial infarction (AMI) were treated with either saruplase (80 mg/hour, n = 236) or alteplase (100 mg every 3 hours, n = 237). Comedication included heparin and acetylsalicylic acid. Angiography was performed at 45 and 60 minutes after the start of thrombolytic therapy. When flow was insufficient, angiography was repeated at 90 minutes. Coronary angioplasty was then performed if Thrombolysis In Myocardial Infarction (TIMI) trial 0 to 1 flow was seen. Control angiography was at 24 to 40 hours. Baseline characteristics were similar. Angiography showed comparable and remarkably high early patency rates (TIMI 2 or 3 flow) in both treatment groups: at 45 minutes, 74.6% versus 68.9% (p = 0.22); and at 60 minutes 79.9% versus 75.3% (p = 0.26). Patency rates at 90 minutes before additional interventions were also comparable (79.9% and 81.4%). Angiographic reocclusion rates were not significantly different: 1.2% versus 2.4% (p = 0.68). After rescue angioplasty, angiographic reocclusion rates of 22.0% and 15.0% were observed. Safety data were similar for both groups. Thus, (1) early patency rates were high for saruplase and alteplase treatment, (2) reocclusion rates for both drugs were remarkably low, and (3) complication rates were similar. Thus, saruplase seems to be as safe and effective as alteplase.

The Inhibition of Endothelial Activation by Unsaturated Fatty Acids

Dietary long-chain fatty acids (FA) may influence pathological processes involving endothelial activation and leukocyte-endothelial interactions, such as inflammation and atherosclerosis. We previously showed that the n-3 FA docosahexaenoate (22:6n-3, DHA) inhibits cytokine-stimulated expression of endothelial-leukocyte adhesion molecules and soluble cytokines in the range of nutritionally achievable plasma concentrations. More recently we assessed structural determinants of VCAM-1 inhibition by FA. Cultured endothelial cells were incubated first with various saturated, monounsaturated, n-6 or n-3 polyunsaturated FA alone and then together with interleukin-1 or tumor necrosis factor. Saturated FA did not inhibit cytokine-induced endothelial activation, while a progressive increase in inhibitory activity was observed, for the same chain length, with the increase in double bonds accompanying the transition from monounsaturates to n-6 and, further, to n-3 FA. Comparison of various FA indicated no role of the double-bond position or configuration; the greater number of double bonds could explain the greater inhibitory activity of n-3 vs. n-6 FA. In order to ascertain mechanisms for these effects, we demonstrated inhibition of nuclear factor-κB (NF-κB) activation by DHA in parallel with a reduction in hydrogen peroxide (a critical mediator of NF-κB activation) released by endothelial cells either extracellularly or intracellularly. This suggests that a property related to fatty acid peroxidability (the presence of multiple double bonds) is related to inhibitory properties of hydrogen peroxide release and, consequently, of endothelial activation.

Quantification of mitral valve stenosis by three-dimensional transesophageal echocardiography

The aim of this study was the evaluation of the diagnostic potentials of transesophageal 3D-echocardiography in the determination of mitral valve stenosis. 54 patients were investigated by transthoracic and multiplane transesophageal echocardiography. In 41 patients cardiac catheterization was performed. 3D-echocardiographic data acquisition was performed by automatic transducer rotation at 2° increments over a span of 180°. The transesophageal probe was linked to an ultrasound unit and to a 3D-workstation capable of ECG- and respiration gated data acquisition, postprocessing and 2D/ 3D image reconstruction. The mitral valve was visualized in sequential cross-sectional planes out of the 3D data set. The spatial position of the planes was indicated in a reference image. In the cross-sectional plane with the narrowest part of the leaflets the orifice area was measured by planimetry. For topographic information a 3D view down from the top of the left atrium was reconstructed. Measurements were compared to conventional transthoracic planimetry, to Doppler-echocardiographic pressure half time and to invasive data. The mean difference to transthoracic planimetry, pressure half time and to invasive measurements were 0.3 ± 0.1 cm2, 0.2 ± 0.1 cm2 and 0.1 ± 0.1 cm2, respectively. Remarkable differences between the 3D- echocardiographic and the 2D- or Doppler- echocardiographic methods were observed in patients with severe calcification or aortic regurgitation. In 22% of the patients the 3D data set was not of diagnostic quality. New diagnostic information from a 3D view of the mitral valve could be obtained in 69% of the patients. Thus, although image quality is limited, 3D- echocardiography provides new topographic information in mitral valve stenosis. It allows the use of a new quantitative method, by which image plane positioning errors and flow-dependent calculation is avoided.

The CYP2J2 G-50T polymorphism and myocardial infarction in patients with cardiovascular risk profile

BackgroundCytochrome P450 (CYP) enzyme 2J2, an epoxygenase predominantly expressed in the heart, metabolises arachidonic acid to biologically active eicosanoids. One of the CYP2J2 products, 11, 12-epoxyeicosatrienoic acid, has several vasoprotective effects. The CYP2J2-G-50T-promotor polymorphism decreases gene expression and is associated with coronary artery disease. This association supports the vascular protective role of CYP-derived eicosanoids in cardiovascular disease. In the present study, we investigated the influence of this polymorphism on survived myocardial infarction in two study groups of patients with on average high cardiovascular risk profile.MethodsThe CYP2J2 polymorphism was genotyped in two groups of patients that were collected with the same method of clinical data collection. Data from 512 patients with sleep apnoea (group: OSA) and on average high cardiovascular risk profile and from another 488 patients who were admitted for coronary angiography (CAR-group) were evaluated for a potential correlation of the CYP2J2 polymorphism G-50T and a history of myocardial infarction. The G-50T polymorphism of the CYP2J2 gene was genotyped by allele specific restriction and light cycler analysis.ResultsThe T-allele of the polymorphism was found in 111 (11.1%; CAR-group: N = 65, 13.3%; OSA: N = 46, 9.0%). 146 patients had a history of myocardial infarction (CAR: N = 120, 24.6%; OSA: N = 26, 5.1%). Cardiovascular risk factors were equally distributed between the different genotypes of the CYP2J2 G-50T polymorphism. In the total group of 1000 individuals, carriers of the T-allele had significantly more myocardial infarctions compared to carriers of the wild type (T/T or G/T: 21.6%; G/G: 13.7%; p = 0.026, odds ratio 1.73, 95%-CI [1.06–2.83]). In the multivariate logistic regression analysis the odds ratio for a history of myocardial infarction in carriers of the T-allele was 1.611, 95%-CI [0.957–2.731] but this trend was not significant (p = 0.073).ConclusionIn presence of other risk factors, the CYP2J2 G-50T failed to show a significant role in the development of myocardial infarction. However, since our result is close to the border of significance, this question should be clarified in larger, prospective studies in the future.