Otmar Pachinger

HMG-CoA Reductase Inhibitors Regulate Inflammatory Transcription Factors in Human Endothelial and Vascular Smooth Muscle Cells

Objective—Pleiotropic atheroprotective effects of HMG-CoA reductase inhibitors may be mediated on the level of vascular gene transcription. The aim of this study was to characterize the effects of statins on the activation of transcription factors known to regulate inflammation and cell proliferation/differentiation. Methods and Results—Simvastatin, atorvastatin, and lovastatin (0.1 to 10 &mgr;mol/L) inhibited the binding of nuclear proteins to both the nuclear factor-kappa B (NF-&kgr;B) and activator protein-1 (AP-1) DNA consensus oligonucleotides in human endothelial and vascular smooth muscle cells as assessed by electrophoretic mobility shift assay (EMSA). The inhibitory effects of statins on NF-&kgr;B or AP-1–dependent transcriptional activity were examined by transient transfection studies. HMG-CoA reductase inhibitors upregulated I&kgr;B-&agr; protein levels in endothelial cells and decreased c-Jun mRNA expression in smooth muscle cells as analyzed by Western and Northern blotting, respectively. Furthermore, statins inhibited DNA binding of hypoxia-inducible factor-1&agr;. Downstream effects of statins included inhibition of plasminogen activator inhibitor-1 and vascular endothelial growth factor-A mRNA levels in endothelial cells. Conclusions—HMG-CoA reductase inhibitors downregulate the activation of transcription factors NF-&kgr;B, AP-1, and hypoxia-inducible factor-1&agr;. These findings support the concept that statins have antiinflammatory and antiproliferative effects that are relevant in the treatment of atherosclerotic diseases.

Endothelial Dysfunction Induced by Hyperhomocyst(e)inemia Role of Asymmetric Dimethylarginine

Background—Endothelial function is impaired by hyperhomocyst(e)inemia. We have previously shown that homocyst(e)ine (Hcy) inhibits NO production by cultured endothelial cells by causing the accumulation of asymmetric dimethylarginine (ADMA). The present study was designed to determine if the same mechanism is operative in humans. Methods and Results—We studied 9 patients with documented peripheral arterial disease (6 men; 3 women; age, 64±3 years), 9 age-matched individuals at risk for atherosclerosis (older adults; 9 men; age, 65±1 years), and 5 young control subjects (younger adults; 5 men; age, 31±1 years) without evidence of or risk factors for atherosclerosis. Endothelial function was measured by flow-mediated vasodilatation of the brachial artery before and 4 hours after a methionine-loading test (100 mg/kg body weight, administered orally). In addition, blood was drawn at both time points for measurements of Hcy and ADMA concentrations. Plasma Hcy increased after the methionine-loading test in each group (all, P <0.001). Plasma ADMA levels rose in all subjects, from 0.9±0.2 to 1.6±0.2 &mgr;mol/L in younger adults, from 1.5±0.2 to 3.0±0.4 &mgr;mol/L in older adults, and from 1.8±0.1 to 3.9±0.3 &mgr;mol/L in peripheral arterial disease patients (all, P <0.001). Flow-mediated vasodilatation was reduced from 13±2% to 10±1% in younger adults, from 6±1% to 5±1% in older adults, and from 7±1% to 3±1% in peripheral arterial disease patients (all, P <0.001). Furthermore, we found positive correlations between plasma Hcy and ADMA concentrations (P =0.03, r =0.450), as well as ADMA and flow-mediated vasodilatation (P =0.002, r =0.623). Conclusions—Our results suggest that experimental hyperhomocyst(e)inemia leads to accumulation of the endogenous NO synthase inhibitor ADMA, accompanied by varying degrees of endothelial dysfunction according to the preexisting state of cardiovascular health.

Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction

Brain natriuretic peptide (BNP), NT-proBNP and NT-pro-atrial natriuretic peptide (NT-proANP) were measured in blood samples from 57 patients using immunoassays and immunoradiometric assays to evaluate the usefulness as diagnostic markers for the detection of heart failure. For the detection of impaired left ventricular ejection fraction (LVEF), receiver operating characteristic curves showed that BNP had the best diagnostic performance with an area under curve (AUC) of 0.75+/-0.06. However, NT-proBNP (AUC: 0.67+/-0.07) and NT-proANP (AUC: 0.69+/-0.08) showed no significant difference to BNP. In a further analysis for the detection of resting LVEF <40%, BNP again was the best marker with an AUC of 0.83+/-0.06. NT-proBNP showed only a slightly smaller AUC (0.79+/-0.07). The AUC for NT-proANP was significantly smaller (0.65+/-0.08) compared to BNP. Additionally, BNP and NT-proBNP correlated negatively with the resting LVEF (BNP: -0.472, p<0.001; NT-proBNP: -0.306, p=0.026), whereas NT-proANP showed no significant correlation. In summary, BNP was the best marker to detect patients with impaired LVEF compared to NT-proBNP and NT-proANP. However, NT-proBNP showed no significant differences to BNP and it is therefore a new promising alternative marker for the detection of left ventricular dysfunction.

Heme oxygenase activity modulates vascular endothelial growth factor synthesis in vascular smooth muscle cells.

Hypoxia, cytokines, and nitric oxide (NO) stimulate the generation of vascular endothelial growth factor (VEGF) and induce heme oxygenase-1 (HO-1) expression in vascular tissue. HO-1 degrades heme to carbon monoxide (CO), iron, and biliverdin, the latter being reduced to bilirubin by biliverdin reductase. In the present study, we investigated the role of HO-1 in the modulation of VEGF synthesis in rat vascular smooth muscle cells (VSMC). In VSMC stimulated with cytokines, inhibition of NO production significantly, but not completely, reduced VEGF release. In contrast, inhibition of HO activity by tin protoporphyrin IX (SnPPIX) totally prevented cytokine-induced increase in VEGF, despite an augmented synthesis of intracellular NO. Stimulation of HO-1 activity by hemin enhanced VEGF production; this effect was abrogated by blockade of the HO pathway. Similarly, VEGF synthesis induced by hypoxia was down-regulated by SnPPIX, but not by inhibitors of NO synthase. To elucidate further a direct involvement of HO-1 in the observed effects, we generated transfected cells that overexpressed the HO-1 gene. Notably, these cells synthesized significantly more VEGF protein than cells transfected with a control gene. Among the products of HO-1, biliverdin and bilirubin showed no effect, whereas iron ions inhibited VEGF synthesis. Exposure of cells to 1% CO resulted in a marked accumulation of VEGF (20-fold increase) over the basal level. Our data indicate that HO-1 activity influences the generation of VEGF in VSMC in both normoxic and hypoxic conditions. As CO and iron, respectively the inducer and the inhibitor of VEGF synthesis, are concomitantly produced during the degradation of heme, these data indicate that HO by-products may differentially modulate VEGF production.

Genetic augmentation of nitric oxide synthase increases the vascular generation of VEGF

OBJECTIVE Vascular endothelial growth factor (VEGF) induces the release of nitric oxide (NO) from endothelial cells. There is also limited data suggesting that NO may enhance VEGF generation. METHODS To further investigate this interaction, we examined the effect of exogenous and endogenous NO on the synthesis of VEGF by rat and human vascular smooth muscle cells (VSMC) by exposing cells to exogenous NO donors, or to genetic augmentation of eNOS or iNOS. RESULTS NO-donors potentiated by 2-fold the generation of VEGF protein by rat or human VSMC. Similarly, rat or human VSMC transiently transfected with plasmid DNA encoding eNOS or iNOS, synthesized up to 3-fold more VEGF than those transfected with control plasmid DNA, an effect which was reversed after treatment with the NOS antagonist L-NAME. Rat VSMC stably transfected with pKeNOS plasmid, constitutively produced NO and released high concentrations of VEGF. In these cells, L-NAME significantly reduced NO synthesis and decreased VEGF generation. The VEGF protein produced by NOS-transfected VSMC was biologically active, as conditioned media harvested from these cells increased endothelial cell proliferation. CONCLUSION These studies reveal that NO derived from NO-donors or generated by NOS within the cells, upregulates the synthesis of VEGF in vascular smooth muscle cells. Administration of NO donors, or augmentation of endogenous NO synthesis, may be an alternative approach in therapeutic angiogenesis.

Sixty-four slice CT evaluation of aortic stenosis using planimetry of the aortic valve area.

OBJECTIVE The purpose of our study was to evaluate planimetry of the aortic valve area with 64-slice CT in comparison with transthoracic echocardiography (TTE) and transesophageal echocardiography (TEE) in patients with aortic stenosis. MATERIALS AND METHODS Thirty-six patients with aortic valve disease referred for coronary 64-slice CT angiography were examined. Planimetry of the aortic valve area with 64-slice CT was compared with TTE using the Doppler continuity equation for calculation of the aortic valve area and with planimetric measurement of the aortic valve area using TEE. RESULTS Planimetry of the aortic valve area with CT (1.11 +/- 0.42 cm2) showed a good correlation with TTE (1.05 +/- 0.42 cm2) (r = 0.88, p < 0.001) in 32 patients and a good correlation with TEE (1.41 +/- 1.61 cm2) (r = 0.99, p < 0.0001) in 10 patients. The mean and maximum transvalvular pressure gradients were correlated with the aortic valve area as measured with CT (r = -0.68, p = 0.0001; and r = -0.67, p = 0.0001, respectively). Beta-blockers were not given (mean heart rate, 62.5 +/- 10.7 beats per minute). CONCLUSION MDCT allows accurate planimetry of the aortic valve area in patients with aortic stenosis. In patients referred for 64-slice CT coronary angiography, concomitant aortic stenosis can be identified and evaluated.

Simultaneous Hybrid COronary Revascularization Using Totally Endoscopic Left Internal Mammary Artery Bypass Grafting and Placement of RapamycIN Eluting Stents in the SAme IntervenTIONal Session

Objectives: Hybrid coronary revascularization procedures apply minimally invasive coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) for treatment of multivessel coronary artery disease. For logistic reasons simultaneous procedures would be desirable. In a pilot study the feasibility of simultaneous robotic totally endoscopic CABG and PCI using drug eluting stents was assessed. Patients and Methods: Five patients were scheduled to undergo simultaneous combined coronary inter- vention. A left internal mammary artery bypass graft was placed to the left anterior descending artery (LAD) in a completely endoscopic fashion using the daVinciTM telemanipulation system. PCI was carried out in the surgical operating room with the GE OEC9800 mobile coronary angiography C-arm. Rapamycin coated Cypher™ stents were placed into stenotic non-LAD targets. Results: The procedure was feasible in 4 patients, one patient was converted to a double CABG operation. There were no significant postoperative clinical complications and patients were discharged from intensive care unit and the hospital after 19 (18–61) hours and 6 (5–7) days respectively. At 6 months postoperatively all patients are free from angina. Conclusion: We conclude that simultaneous robotic totally endoscopic left internal mammary artery to LAD placement and PCI to non-LAD targets using drug eluting stents is feasible in one session.

Low apolipoprotein A-IV plasma concentrations in men with coronary artery disease

OBJECTIVES The objective of this study was to evaluate the relation between apolipoprotein A-IV (apoA-IV) plasma concentrations and coronary artery disease (CAD). BACKGROUND Experimental in vitro and in vivo studies favor apoA-IV to be protective against the development of atherosclerosis. Mice that overexpress either human or mouse apoA-IV demonstrated a significant reduction of aortic atherosclerotic lesions compared with control mice. Data on apoA-IV plasma concentrations and CAD in humans are lacking. METHODS We determined in two independent case-control studies of a Caucasian and an Asian Indian population whether apoA-IV plasma concentrations are related to the presence of angiographically assessed CAD. RESULTS Plasma apoA-IV levels were significantly lower in 114 male Caucasian subjects with angiographically defined CAD when compared with 114 age-adjusted male controls (10.2 +/-3.8 mg/dL vs. 15.1 +/- 4.0 mg/dL, p < 0.001). Logistic regression analysis indicated that the association between apoA-IV levels and CAD was independent of the high-density lipoprotein cholesterol and triglyceride concentrations. The inverse relationship between plasma levels of apoA-IV and the presence of CAD was confirmed in an independent sample of 68 male Asian Indians with angiographically documented CAD and 68 age-matched controls. CONCLUSIONS The results of this cross-sectional study demonstrate for the first time an association between low apoA-IV concentrations and CAD in humans and suggest that apoA-IV may play an antiatherogenic role in humans.

Statins differentially regulate vascular endothelial growth factor synthesis in endothelial and vascular smooth muscle cells

OBJECTIVES HMG-CoA reductase inhibitors (statins) can modulate the formation of new blood vessels, but the reports on their contribution to angiogenesis are contradictory. Therefore, we investigated whether the effect of statins is dependent either on the concentration of the drug or on the cell type. METHODS AND RESULTS Under basal conditions human vascular smooth muscle cells (HVSMC) and microvascular endothelial cells (HMEC-1) constitutively generate and release vascular endothelial growth factor (VEGF). In contrast, primary macrovascular endothelial cells (HUVEC) produce minute amounts of VEGF. Different statins (atorvastatin, simvastatin and lovastatin, 1-10 micromol/l) significantly reduced basal and cytokine-, nitric oxide- or lysophosphatidylcholine (LPC)-induced VEGF synthesis in HMEC-1 and HVSMC. Interestingly, at the same concentrations statins upregulated VEGF generation in HUVEC. Furthermore, statins exerted dual, concentration-dependent influence on angiogenic activities of HUVEC as determined by tube formation assay. At low concentrations (0.03-1 micromol/l) the pro-angiogenic activity of statins is prevalent, whereas at higher concentrations statins inhibit angiogenesis, despite increasing VEGF synthesis. CONCLUSION Our data show that statins exert concentration- and cell type-dependent effects on angiogenic activity of endothelial cells and on VEGF synthesis. The data are of relevance for elucidating the differential activity of statins on angiogenesis in cardiovascular diseases and cancer.

Relation of coronary arterial occlusion pressure during percutaneous transluminal coronary angioplasty to presence of collaterals

To investigate the relation of the gradient across a coronary artery stenosis and the pressure distal to the stenosis after proximal occlusion during percutaneous transluminal coronary angioplasty to the amount of angiographically estimated collateral circulation, 63 patients (55 men, 8 women) were studied. All patients had 1-vessel disease (54 left anterior descending, 8 right coronary artery and 1 circumflex coronary artery). All patients had documented ischemia, and angioplasty was carried out within 4 weeks after the initial angiogram. The patients were separated into 4 groups: 0 = no collaterals (35 patients), +1 = just visible collaterals (8 patients), +2 = collaterals without reaching the contralateral vessel (10 patients), and +3 = filling of the contralateral vessel (10 patients). There was no difference in age among the 4 groups. There was a significant negative relation of the gradient vs the extent of collateral circulation, although the degree of stenosis increased significantly from group 0 to group +3. There was a significant positive relation of the occlusion pressure (in absolute terms and in percent of the proximal systolic pressure) vs the extent of collateral circulation. There was a significantly smaller change of the occlusion pressure vs the distal pressure before occlusion if good collaterals were present. The occlusion pressure remained constant during 1 occlusion up to 40 seconds and was reproducible in 3 successive occlusions. In conclusion, the pressure distal to a coronary artery stenosis is mainly dependent on the severity of the stenosis and on the collateral flow. If anterograde flow is eliminated by proximal occlusion the distal pressure is only dependent on the extent of collateral circulation.