Christiane P. Tiefenbacher

Endothelial Dysfunction of Coronary Resistance Arteries Is Improved by Tetrahydrobiopterin in Atherosclerosis

BackgroundTetrahydrobiopterin (BH4), an essential cofactor for the synthesis of NO, improves endothelial dysfunction after ischemia/reperfusion. Therefore, we hypothesized that reduction of BH4 is involved in the attenuation of endothelium-dependent vasodilation in atherosclerosis, and we investigated the effect of alterations of the BH4 level on the vasodilatory potential of coronary resistance vessels from humans and pigs with atherosclerosis. Methods and ResultsCoronary arterioles were obtained from patients undergoing CABG (atherosclerosis group) or valve replacement (control group) and from pigs fed either a standard diet (control group) or atherogenic diet (atherosclerosis group). After isolation, vessels were cannulated, pressurized, and placed on the stage of an inverted microscope. Dose-response curves were investigated in response to the endothelium-dependent agonists histamine, serotonin, and acetylcholine (for pigs, substance P) and to the endothelium-independent agonist sodium nitroprusside (SNP) under control conditions and before and after incubation of the vessels with sepiapterin (substrate for BH4 synthesis). In vessels from patients and from animals with atherosclerosis, compared with vessels from the control groups, there was a significant (P <0.05) reduction of vasodilation to all tested endothelium-dependent agonists but not to SNP. After application of sepiapterin, the responses to the endothelium-dependent agonists but not to SNP were significantly improved in vessels from the atherosclerosis groups. Sepiapterin did not influence vascular reactivity in the control groups. ConclusionsAtherosclerosis severely compromises endothelial function of coronary resistance arteries. Administration of sepiapterin leads to a significant improvement of endothelium-dependent vasodilatation to different agonists in vessels from humans and pigs with atherosclerosis. Therefore, we conclude that a reduced availability of BH4 is involved in the development of endothelial dysfunction in atherosclerosis.

Transcatheter Implantation of the MONARC Coronary Sinus Device for Mitral Regurgitation 1-Year Results From the EVOLUTION Phase I Study (Clinical Evaluation of the Edwards Lifesciences Percutaneous Mitral Annuloplasty System for The Treatment of Mitral Regurgitation)

OBJECTIVES This study sought to assess the safety and efficacy of transcatheter valve annuloplasty in patients with mitral regurgitation (MR). BACKGROUND Mitral regurgitation is associated with a worsened prognosis in patients with dilated cardiomyopathy. Surgical mitral annuloplasty reduces the septal-lateral dimension of the mitral annulus resulting in improved leaflet coaptation with a reduction in regurgitation. Percutaneous annuloplasty with the MONARC device (Edwards Lifesciences, Irvine, California) implanted within the coronary sinus is designed to reduce mitral regurgitation through a similar mechanism. METHODS A total of 72 patients with MR grade ≥ 2 were enrolled at 8 participating centers in 4 countries. Clinical evaluation and transthoracic echocardiography were performed at baseline and at 3, 6, and 12 months. Multislice cardiac computed tomography and coronary angiography were performed at baseline and 3 months. RESULTS The MONARC device was implanted in 59 of 72 patients (82%). The primary safety end point (freedom from death, tamponade, or myocardial infarction at 30 days) was met in 91% of patients at 30 days and in 82% at 1 year. Computed tomography imaging documented passage of the great cardiac vein over an obtuse marginal artery in 55% of patients and was associated with angiographic coronary artery compression in 15 patients and myocardial infarction in 2 patients (3.4%). At 12 months, a reduction in MR by ≥ 1 grade was observed in 50.0% of 22 implanted patients with matched echocardiograms and in 85.7% of 7 patients with baseline MR grade ≥ 3. CONCLUSIONS Implantation of the MONARC device in the coronary sinus is feasible and may reduce MR. However, coronary artery compression may occur in patients in whom the great cardiac vein passes over a coronary artery, necessitating strategies in future studies to avoid this occurrence.

Myocardial preconditioning and remote renal preconditioning--identifying a protective factor using proteomic methods?

AbstractIt is still unknown whether remote ischemic preconditioning is mediated by a humoral or a neurogenic mechanism from the preconditioning to the preconditioned tissue. The purpose of the following study was to identify a possible humoral trigger of ischemic myocardial preconditioning and remote renal preconditioning. Open chest rats were subjected to a coronary artery occlusion period of 45 min followed by 2 h of reperfusion (Control animals; n = 6). The coronary preconditioned group (IPC, n = 6) was subjected to a preceding preconditioning period of 5 min coronary artery occlusion followed by 5 min of reperfusion, repeated three times. The renal preconditioned group (IPR, n = 6) was subjected to a preceding renal artery occlusion period of 10 min followed by 20 min of reperfusion. Area at risk (AAR) and infarcted area (IA) were determined at the end of each protocol. Blood samples were taken at the end of the preconditioning protocols from parallel experiments for proteomic analysis using two–dimensional gel electrophoresis (2-DE), matrix assisted laser desorption and ionization—time of flight—mass spectrometry (MALDI–TOF–MS), and liquid chromatography—electrospray ionization—tandem mass spectrometry (nanoLC–ESI–MS/MS). IA/AAR was 87.8 ± 10.7% in the control group. IPC and IPR signi.cantly reduced IA/AAR (58.2 ± 9.3% and 56.9 ± 9.0%, p < 0.001). Proteomic analyses detected four protein spots which were either up– (n = 3) or down–regulated in the preconditioned groups vs. the control group. The three up–regulated protein spots were identi.ed as albumin fragments, whereas the downregulated spot was identified as liver regeneration–related protein (LRRG03). Interestingly, albumin modification by brief ischemia has been recently shown and evaluated for the clinical diagnosis of sublethal myocardial ischemia. However, no differentially abundant proteins which possess a known signaling function could be found. Hence, though there is a differential protein expression in blood following IPC and IPR, our data are not in favor of a humoral mediator of remote preconditioning with a molecular weight of more than 8 kDa. Our results rather suggest either a neurogenic pathway or a mediator smaller than 8 kDa.

Increased Infarct Size in Uremic Rats: Reduced Ischemia Tolerance?

In patients with renal failure, myocardial infarction (MI) is more frequent and the rate of death from acute MI is very high. It has been argued that ischemia tolerance of the heart is reduced in uremia, but direct evidence for this hypothesis has not been provided. It was the purpose of this study (1) to ligate the left coronary artery and to measure the nonperfused area (risk area: total infarction plus penumbra) as well as the area of total infarction in subtotally nephrectomized (SNX) rats compared with sham-operated pair-fed control rats and (2) to examine the effects of potential confounders such as BP, sympathetic overactivity, and salt retention. The left coronary artery was ligated for 60 min, followed by reperfusion for 90 min. For visualizing perfused myocardium, lissamine green ink was injected. The nonperfused area (lissamine exclusion) and the area of total infarction (triphenyltetrazolium chloride stain) were assessed in sections of the left ventricle using image analysis. Groups of SNX rats also received: antihypertensive treatment (nadolol plus hydralazine); moxonidine; high salt diet or low salt diet (1.58% versus 0.015%). In surviving animals, the nonperfused area at risk (as the proportion of total left ventricular area), presumably determined by the geometry of vascular supply, was similar in sham-operated and SNX animals (0.38 +/- 0.13 versus 0.45 +/- 0.09; NS). In contrast, the infarcted area, given as a proportion of the nonperfused risk area, was significantly (P < 0.003) higher in SNX (0.68 +/- 0.09) compared with sham-operated (0.51 +/- 0.11) rats and was not altered by any of the above interventions. The finding that a greater proportion of nonperfused myocardium undergoes total necrosis is consistent with the hypothesis of reduced ischemia tolerance of the heart in renal failure. The findings could explain the high rate of death from MI in patients with impaired renal function.

Expression of nitric oxide related enzymes in coronary heart disease

Enzymes involved in the metabolism nitric oxide (NO) and reactive oxygen species (ROS) may play a role for the decreased availability of NO in atherosclerosis. We, therefore, hypothesized that the pattern of gene expression of these enzymes is altered in atherosclerosis. Myocardial tissue from patients with coronary heart disease (CHD) or without CHD (control group) was investigated. The level of enzymes related to NO/ROS metabolism was determined both at mRNA level and protein level by rt-PCR, real-time PCR, and western blot. The expression of NOS1–3 (synthesis of NO), arginase1 (reduction of l-arginine), p22phox (active subunit of NADPH oxidase), GTPCH (rate limiting enzyme for tetrahydrobiopterin), SOD1–3 (scavengers of superoxide anions), PRTMT1–3, and DDAH2 (involved in the metabolism of ADMA) was determined. All enzymes were found to be expressed in human myocardium. NOS isoforms were decreased in CHD in protein level, but only the downregulation of NOS3 expression reached statistical significance. The expression of PRMT1 and PRMT3 was increased. In addition, the expression of DDAH2 was reduced, both theoretically leading to an increase of ADMA concentration. SOD3 was downregulated in tissue from patients with CHD. Taken together, in myocardial tissue from patients with atherosclerosis, the expression of genes increasing ADMA levels is enhanced in contrast to a reduced expression of genes promoting NO synthesis. These results may contribute to the explanation of increased oxidative stress in atherosclerosis on the level of gene expression.

Basic fibroblast growth factor and heparin influence coronary arteriolar tone by causing endothelium-dependent dilation

OBJECTIVE The strong angiogenic and mitogenic agents acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) share signalling pathways with known vasodilatory agonists. Therefore, we hypothesized the FGFs produce vasoactive responses. We also proposed that heparin would exert a similar action to FGF, because this proteoglycan not only binds to the FGF receptor, but also facilitates the release of FGF from the cardiac extracellular matrix and promotes its binding to a high-affinity receptor. To test these hypotheses, we examined the vasodilatory reactions of coronary arterioles to aFGF, bFGF, and heparin, and the effects of antagonists to nitric oxide synthase (L-NMMA), prostaglandins (indomethacin), ATP-sensitive potassium (K[ATP]) channels (glibenclamide), FGF and FGF receptors on the vasoactive responses. METHODS Arterioles (70-110 microns, internal diameter) were dissected from pig hearts and cannulated with micropipettes. Diameter was determined with videomicroscopy in response to bFGF and aFGF in concentrations of 1-100 ng/ml and to heparin (5-200 U/ml). RESULTS Basic FGF, but not aFGF, caused dose-dependent vasodilation with a maximum of 61 +/- 4%. Relaxation of bFGF was antagonized by pretreatment with L-NMMA, but was not affected by pretreatment with indomethacin or glibenclamide. Heparin caused dose-dependent vasodilation with a maximum of 100 +/- 3% which was partially blocked by either L-NMMA or glibenclamide, but not by indomethacin. Furthermore, the effect of bFGF could be significantly blocked by pretreatment with an FGF receptor antibody as well as with a monoclonal antibody against FGF. Pretreatment with both antibodies significantly inhibited also the effect of heparin. CONCLUSIONS These results indicate that bFGF and heparin cause vasodilation of coronary arterioles via an increase in NO production and heparin additionally by other mechanisms such as by activating K[ATP] channels. Furthermore, the effect of heparin is partially mediated via FGF and FGF receptors. We therefore speculate that both substances may be involved in the regulation of coronary microvascular tone acting partially through the same signalling mechanisms.

Role of human GTP cyclohydrolase I and its regulatory protein in tetrahydrobiopterin metabolism

Abstract.Objective: GTP cyclohydrolase I (GTPCH I) catalyzes the de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of NO-synthase. The enzyme underlies negative feedback regulation by the end product BH4. This feedback inhibition is mediated through complex formation with the GTP cyclohydrolase I feedback regulatory protein (GFRP). To further classify the mechanism involved in the regulation of BH4 synthesis, we measured expression of GTPCH I and GFRP in different human tissues. Furthermore, we looked for the influence of phenylalanine that is known to reverse BH4-mediated feedback inhibition of GTPCH I, and of immunostimulation with interferon γ on the expression of GTPCH I and GFRP. Methods and results: Using RT-PCR and northern blot technique, coexpression of GFRP and GTPCH I could be demonstrated in a number of different tissues such as endothelial cells and peripheral blood cells. Following stimulation of human umbilical vein endothelial cells (HUVEC) with phenylalanine (1 mM), there was no change of GFRP mRNA. In contrast, the mRNA level of GTPCH I was significantly upregulated with a maximum after 6 hours (p = 0.04). Incubation of HUVEC with interferon-γ (100 U/ml) showed an increase of GTPCH I mRNA and a significant downregulation of GFRP mRNA after 24 hours (p = 0.03). Conclusion: This study shows for the first time the expression of GFRP in different human tissues. The biosynthesis of BH4 is not only regulated on the substrate level but also through transcription of the interacting proteins. Phenylalanine stimulates the biosynthesis of BH4 not only by reversing the negative feedback inhibition of GTPCH I but also by increasing the mRNA level of GTPCH I. Immunostimulation alters protein expression of GTPCH I and GFRP in a way that favors BH4 synthesis.

Pathology, natural history and treatment of abdominal aortic aneurysms

SummaryWith increasing age of the population and improvement of diagnostic tools, the incidence of abdominal aortic aneurysms (AAA) has been rising steadily. Despite an improvement in operative and interventional treatment options, AAA is the cause of death in 1–3% of men over 65 years of age in industrial countries, mostly due to rupture [1]. Therefore, routine screening for AAA by ultrasonography has been postulated in the past: a 60 year old man with an abdominal aortic diameter of less than 3 cm has a life-time risk of developing AAA close to zero. However, routine screening has not been found to be cost effective. Despite of the results of two well-designed studies, the limits of AAA qualifying the patient for surgery or intervention in contrast to conservative treatment is still a matter of debate. The present review article summarizes the current knowledge of the pathology, incidence, risks, natural course as well as symptoms and current treatment strategies of AAA on the basis of the recent literature.

INHIBITION OF ELASTASE IMPROVES MYOCARDIAL FUNCTION AFTER REPETITIVE ISCHAEMIA AND MYOCARDIAL INFARCTION IN THE RAT HEART

Abstract We investigated the potential of inhibition of elastase, a granulocyte-derived proteolytic enzyme, in ameliorating the effects of myocardial stunning caused by repetitive ischaemia (RI) and myocardial infarction (MI) for the first time in an in situ, perfused, rat heart model. The effects of the elastase-inhibitors Elafin (EL, 10 mg/kg/h) and ICI 200,880 (ICI,5 mg/kg/h) on myocardial blood flow (MBF, H2 clearance), regional myocardial function (FT, pulsed doppler) and neutrophil extravasation (myocardial myeloperoxidase activity, MPO) were investigated in RI (5×10 min ligature of the anterior descending ramus (LAD), 5×20 min reperfusion) and MI (50 min LAD ligature, 60 min reperfusion). Under control conditions, MBF and FT were significantly reduced and MPO was significantly increased after RI (n=8) and MI (n=8) in the ischaemic area compared with baseline. Pretreatment with EL (n=7) or ICI (n=7) did not improve MBF significantly and did not influence the successive attenuation of peak values of reactive hyperaemia. However, both EL and ICI significantly improved FT and significantly reduced MPO after RI and MI compared with control conditions. Additionally, both the area at risk and MI size were reduced significantly by both inhibitors. These results demonstrate that elastase inhibitors significantly improve the reduction of FT both in myocardial stunning and in myocardial infarction in the rat without significant improvement of MBF. It is concluded that elastase inhibitors exert a cardioprotective effect against reperfusion injury, probably by inhibition of leukocyte extravasation as indicated by the decrease in MPO activity.

Heterogeneity of coronary vasomotion

Abstract Apart from local heterogeneities of myocardial blood flow, the regulation of vascular tone is equally complex. Changes in vascular tone are essential for the adaptation of coronary blood flow to varying metabolic demands. The majority of coronary vascular resistance is found in the microcirculation, that is, in vessels with less than 150–200 μm in diameter; therefore, understanding the regulatory mechanisms that exercise control of the tone of these vessels is paramount to our understanding the control of myocardial perfusion. Recently, it became evident that different regulatory systems such as metabolic, neurohumoral, myogenic, and flow-mediated mechanisms have preferential effects on particular microvascular segments. However, the significance of specific vasoactive mediators is still under investigation. Growth factors, which are synthesized in cells in the surrounding of vessels such as mast cells and cardiac myocytes, have recently been suggested to play a role in the coordination of endothelium-dependent and endothelium-independent vasoactive mechanisms. The aim of this review is, first, to focus on the heterogeneity of the regulation of coronary vascular resistance in general and, second, to discuss recent studies showing a possible role of growth factors, especially fibroblast growth factor (FGF), heparin, and endothelin, as modulators of microvascular tone.