Association of circulating factor seven activating protease (FSAP) and of oral Omega-3 fatty acids supplements with clinical outcome in patients with atrial fibrillation: the OMEGA-AF study.

Factor VII Activating Protease (FSAP) activates factor VII (FVII) as well as pro-urokinase (uPA). Our goal was to evaluate the relation between plasma levels of FSAP and clinical instability in atrial fibrillation (AF) and possible effects of oral omega-3 fatty acids (FA) supplements. 101 patients with persistent AF were analyzed in the OMEGA-AF Study. Plasma FSAP levels were measured at baseline and after 12 weeks of treatment with omega-3 FA. The median FSAP antigen concentration, in contrast to FSAP activity, was higher in patients with persistent AF. The maintenance of SR after successful cardioversion (CV) did not lead to a normalization of FSAP concentration. Supplementation with omega-3 FA but not placebo significantly reduced elevated FSAP concentration. Furthermore, elevated FSAP levels did not indicate a significantly increased risk of recurrence of AF after electrical CV or cardiovascular clinical events during 1 year of follow-up. Plasma FSAP concentration was increased in patients with AF and may be involved in the pathogenesis of this condition. The possible effects of omega-3 FA on clinical AF potential could be linked with modulation of circulating FSAP levels.

The G534E polymorphism of the gene encoding the factor VII-activating protease is associated with cardiovascular risk due to increased neointima formation.

The G534E polymorphism (Marburg I [MI]) of factor VII-activating protease (FSAP) is associated with carotid stenosis and cardiovascular disease. We have previously demonstrated that FSAP is present in atherosclerotic plaques and it is a potent inhibitor of vascular smooth muscle proliferation and migration in vitro. The effect of wild-type (WT)- and MI-FSAP on neointima formation in the mouse femoral artery after wire-induced injury was investigated. Local application of WT-FSAP led to a 70% reduction in the neointima formation, and this effect was dependent on the protease activity of FSAP. MI-FSAP did not inhibit neointima formation in vivo. This is due to a reduced proteolytic activity of MI-FSAP, compared to WT-FSAP, toward platelet-derived growth factor BB, a key mediator of neointima development. The inability of MI-FSAP to inhibit vascular smooth muscle accumulation explains the observed linkage between the MI-polymorphism and increased cardiovascular risk. Hence, FSAP has a protective function in the vasculature, and analysis of MI polymorphism is likely to be clinically relevant in restenosis.

Circulating factor VII activating protease (FSAP) is associated with clinical outcome in acute coronary syndrome.

BACKGROUND: Factor VII activating protease (FSAP) is a circulating serine protease strongly expressed in unstable plaques and may serve as a marker of plaque destabilization. The aim of this study was to examine the relation between plasma concentrations of FSAP and clinical instability and outcome in coronary artery disease (CAD). METHODS AND RESULTS: Circulating FSAP concentration and activity, as well as FSAP mRNA expression in monocytes, were measured in 231 sequential patients who underwent coronary angiography because of stable angina pectoris (n=50), unstable angina pectoris (n=43), or acute myocardial infarction (n=87). FSAP activity, but not FSAP antigen concentration, was elevated in patients with CAD compared with a control group. Elevated FSAP activity (≥1.035 plasma equivalent units [PEU]/ml) indicated a significantly increased risk of death or non-fatal myocardial infarction during 1 year of follow-up as compared with patients with low activity of FSAP (odds ratio 1.895 [95% confidence interval 1.093-3.283]; P=0.023). Furthermore, there were no significant changes in the FSAP expression in monocytes from CAD and control subjects in the basal state but there were differences after stimulation with proinflammatory factors. CONCLUSIONS: Plasma FSAP activity was significantly increased in patients with acute coronary syndrome and may be involved in the pathogenesis of these conditions. High levels of FSAP activity were predictive of adverse events during follow-up, suggesting its potential role in risk stratification and clinical management of CAD patients.

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes.

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

3-Deazaadenosine prevents smooth muscle cell proliferation and neointima formation by interfering with Ras signaling.

3-Deazaadenosine (c3Ado) is a potent inhibitor of S-adenosylhomocysteine hydrolase, which regulates cellular methyltransferase activity. In the present study, we sought to determine the effect of c3Ado on vascular smooth muscle cell (VSMC) function and neointima formation in vivo. c3Ado dose-dependently prevented the proliferation and migration of human coronary VSMCs in vitro. This was accompanied by an increased expression of the cyclin-dependent kinase inhibitors p21(WAF1/Cip1), p27(Kip1), a decreased expression of G(1)/S phase cyclins, and a lack of retinoblastoma protein hyperphosphorylation. In accordance with these findings, fluorescence-activated cell-sorting analysis of propidium iodide-stained cells indicated a cell cycle arrest in the G(0)/G(1) phase. Importantly, c3Ado did not affect the number of viable (trypan blue exclusion) or apoptotic cells (TUNEL). Mechanistically, c3Ado prevented FCS-induced Ras carboxyl methylation and membrane translocation and activity by inhibiting isoprenylcysteine carboxyl methyltransferase and reduced FCS-induced extracellular signal-regulated kinase (ERK)1/2 and Akt phosphorylation in a dose-dependent manner. Conversely, rescuing signal transduction by overexpression of a constitutive active Ras mutant abrogated c3Ado's effect on proliferation. For in vivo studies, the femoral artery of C57BL/6 mice was dilated and mice were fed a diet containing 150 microg of c3Ado per day. c3Ado prevented dilation-induced Ras activation, as well as ERK1/2 and Akt phosphorylation in vivo. At day 21, VSMC proliferation (proliferating-cell nuclear antigen [PCNA]-positive cells), as well as the neointima/media ratio (0.7+/-0.2 versus 1.6+/-0.4; P<0.05) were significantly reduced, without any changes in the number of apoptotic cells. Our data indicate that c3Ado interferes with Ras methylation and function and thereby with mitogenic activation of ERK1/2 and Akt, preventing VSMC cell cycle entry and proliferation and neointima formation in vivo. Thus, therapeutic inhibition of S-adenosylhomocysteine hydrolase by c3Ado may represent a save and effective novel approach to prevent vascular proliferative disease.

Time-course analysis on the differentiation of bone marrow-derived progenitor cells into smooth muscle cells during neointima formation.

OBJECTIVE: Bone marrow-derived progenitor cells have been implicated to contribute to neointima formation, but the time course and extent of their accumulation and differentiation into vascular cells and, most importantly, the long-term contribution of bone marrow-derived progenitor cells to the vascular lesion remain undefined. METHODS AND RESULTS: Wire-induced injury of the femoral artery was performed on chimeric C57BL/6 mice transplanted with bone marrow from transgenic mice expressing enhanced green fluorescence protein, and vessels were harvested at 3 days, 1, 2, 3, 4, 6, and 16 weeks after dilatation (n=8 animals per time point). Using high-resolution microscopy, we unexpectedly found that the expression of smooth muscle cell or endothelial cell markers in enhanced green fluorescence protein positive cells was a very rare event. Indeed, most of the enhanced green fluorescence protein positive cells that accumulated during the acute inflammatory response were identified as monocytes/macrophages, and their number declined at later time points. In contrast, a substantial fraction of highly proliferative stem cell antigen-1 and CD34(+) but enhanced green fluorescence protein negative and thus locally derived cells were detected in the adventitia. CONCLUSIONS: These data provide evidence that the differentiation of bone marrow-derived progenitor cells into smooth muscle cell or endothelial cell lineages seems to be an exceedingly rare event. Moreover, the contribution of bone marrow-derived cells to the cellular compartment of the neointima is limited to a transient period of the inflammatory response.

Insulin stabilizes microvascular endothelial barrier function via phosphatidylinositol 3-kinase/Akt-mediated Rac1 activation.

OBJECTIVE: Insulin is a key regulator of metabolism, but it also confers protective effects on the cardiovascular system. Here, we analyze the mechanism by which insulin stabilizes endothelial barrier function. METHODS AND RESULTS: Insulin reduced basal and antagonized tumor necrosis factor-alpha-induced macromolecule permeability of rat coronary microvascular endothelial monolayers. It also abolished reperfusion-induced vascular leakage in isolated-perfused rat hearts. Insulin induced dephosphorylation of the regulatory myosin light chains, as well as translocation of actin and vascular endothelial (VE)-cadherin to cell borders, indicating a reduction in contractile activation and stabilization of cell adhesion structures. These protective effects were blocked by genistein or Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester (HNMPA-[AM](3)), a pan-tyrosine-kinase or specific insulin-receptor-kinase inhibitor, respectively. Insulin stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and NO production, and it activated Rac1. Inhibition of PI3K/Akt abrogated Rac1 activation and insulin-induced barrier protection, whereas inhibition of the endothelial nitric oxide synthase/soluble guanylyl cyclase pathway partially inhibited them. Inhibition of Rac1 abrogated the assembly of actin at cell borders. Accordingly, it abolished the protective effect of insulin on barrier function of the cultured endothelial monolayer, as well as the intact coronary system of ischemic-reperfused hearts. CONCLUSIONS: Insulin stabilizes endothelial barrier via inactivation of the endothelial contractile machinery and enhancement of cell-cell adhesions. These effects are mediated via PI3K/Akt- and NO/cGMP-induced Rac1 activation.

Randomized comparison of multipolar, duty-cycled, bipolar-unipolar radiofrequency versus conventional catheter ablation for treatment of common atrial flutter.

INTRODUCTION: Radiofrequency (RF) catheter ablation has been established as an effective and curative treatment for atrial flutter (AFL). Approved methods include a drag-and-drop method, as well as a point-by-point ablation technique. The aim of this study was to compare the acute efficacy and procedural efficiency of a multipolar linear ablation catheter with simultaneous energy delivery to multiple catheter electrodes against conventional RF for treatment of AFL. METHODS: Patients presenting to our department with symptomatic, typical AFL were enrolled consecutively and randomized to conventional RF ablation with an 8-mm tip catheter (ConvRF) or a duty-cycled, bipolar-unipolar RF generator delivering power to a hexapolar tip-versatile ablation catheter (T-VAC) group. For both groups, the procedural endpoint was bidirectional cavotricuspid isthmus block. RESULTS: Sixty patients were enrolled, 30 patients each assigned to ConvRF and T-VAC groups. Total procedure time (40.2 ± 15.8 min vs 60.5 ± 12.7 min), energy delivery time (8.5 ± 3.7 min vs 14.7 ± 5.2 min), radiation dose (14.5 ± 3.5 cGy/cm² vs 31.7 ± 12.1 cGy/cm²), and the minimum number of RF applications needed to achieve block (4.2 ± 2.4 vs 8.9 ± 7.2) were significantly lower in the T-VAC group. In 7 patients treated with the T-VAC catheter, bidirectional block was achieved with less than 3 RF applications, versus no patients with conventional RF energy delivery. CONCLUSION: The treatment of typical AFL using a hexapolar catheter with a multipolar, duty-cycled, bipolar-unipolar RF generator offers comparable effectiveness relative to conventional RF while providing improved procedural efficiency.

Inhibition of matrix deposition: a new strategy for prevention of restenosis after balloon angioplasty.

Restenosis after balloon angioplasty or stent placement is characterized by local accumulation of mainly vascular smooth muscle cells and the synthesis of extracellular matrix molecules (ECM).We hypothesized that inhibition of ECM synthesis represents a strategy to prevent trauma-induced neointima formation. Rats were treated with pirfenidone (1 g/kg of body weight orally.), an inhibitor of growth factor-induced collagen synthesis, and subjected to balloon denudation of the carotid artery. Two weeks later, computer-aided morphometry was done and compared with untreated controls (each n = 6). Neointimal proliferative activity was quantified immunohistochemically by counting PCNA-positive nuclei, and collagen deposition was visualized by picrosirius red staining and semi-quantified by Northern blot. Control-injured animals developed marked neointimal thickening within 2 weeks (I/M, mean intima to media ratio: 2.42 +/- 0.15) resulting in an 89.2% luminal narrowing. The neointima mainly consisted of vascular smooth muscle cells embedded in collagen. Neointima formation was strongly reduced when balloon-injured animals had been treated with pirfenidone (I/M ratio 0.22 +/- 0.08, P < 0.001), resulting in a minimal residual narrowing of the lumen (7.9%). I/M ratio did not further increase even after discontinuation of the drug for 14 days (0.35 +/- 0.13). Proliferative activity within the neointima was unaffected by the drug, 4.4% versus 4.8% of neointimal cells stained positive for PCNA in carotid arteries of treated versus untreated animals, respectively. However, picrosirius red staining demonstrated marked attenuation of collagen deposition, a finding that was further confirmed by Northern blot of homogenized vessels. Pirfenidone, currently being investigated clinically for the treatment of various fibrotic diseases, is able to prevent neointimal lesion formation most likely through inhibition of local ECM deposition. Targeting matrix deposition may have an intriguing potential for the prevention of vascular proliferative diseases.

Intracoronary administration of bone marrow-derived progenitor cells improves left ventricular function in patients at risk for adverse remodeling after acute ST-segment elevation myocardial infarction: results of the Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction study (REPAIR-AMI) cardiac magnetic resonance imaging substudy.

BACKGROUND: Serial cardiac magnetic resonance imaging (CMR) is the reference standard for evaluating left ventricular function, wall motion, and infarct size in patients with acute myocardial infarction, as well as remodeling during follow-up. The cardiac CMR substudy of the randomized multicenter REPAIR-AMI trial (Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction study) aimed at gaining insight into postinfarction left ventricular remodeling processes. METHODS: Consecutive patients with ST-segment elevation myocardial infarction and primary percutaneous coronary intervention were enrolled (n = 204) and randomly assigned to either stem cell therapy (bone marrow-derived progenitor cells [BMC]) or placebo after bone marrow aspiration. In the magnetic resonance imaging substudy, 54 patients completed serial CMR (baseline, 4 and 12 months, respectively) after enrollment (27 BMC, 27 placebo). Image analysis was performed at a central core laboratory. RESULTS: There were no significant differences between the 2 groups with respect to global ejection fraction (EF), end-diastolic volume (EDV), and end-systolic volume (ESV) at baseline. At 12 months, the treatment effect of BMC infusion on EF amounted to 2.8 absolute percentage points (P = .26), the progression of EDV at 12 months was less in the BMC group (treatment effect 14 mL, P = .12), and unlike placebo, ESV did not increase (absolute treatment effect 13 mL, P = .08), respectively. In patients with a baseline EF < median (EF < or = 48.9%), BMC administration was associated with a significantly improved EF (+6.6%, P = .01), reduced EDV increase (treatment effect 29.1 mL, P = .02), and abrogation of ESV increase (treatment effect 29.4 mL, P = .01) after 12 months, respectively. CONCLUSION: Intracoronary administration of BMC additionally improved left ventricular function in patients with impaired left ventricular function after ST-segment elevation myocardial infarction despite optimal "state-of-the-art" reperfusion and pharmacologic treatment on 1-year follow-up and beneficially interfered with adverse postinfarction left ventricular remodeling.

Calpain counteracts mechanosensitive apoptosis of vascular smooth muscle cells in vitro and in vivo.

Mechanical forces contribute to vascular remodeling processes. Elevated mechanical stress causes apoptosis of vascular smooth muscle cells (VSMCs) within the media. This study examined the role of the cystein protease calpain in force-induced vascular cell apoptosis and its effect on injury-induced vascular remodeling processes. VSMCs were exposed to cyclic tensile force in vitro, which resulted in increased p53 protein expression and transcriptional activity as well as a significant increase of apoptotic VSMCs. Apoptosis was prevented by the p53 inhibitor pifithrin and by p53 antisense oligonucleotides, indicating dependency of force-induced apoptosis on p53. Simultaneously, calpain activity increased by mechanical stress. Prevention of calpain activation by calpeptin or antisense oligonucleotides augmented strain-induced p53 expression and transcriptional activity, resulting in a further increase of apoptotic rate. p53 protein was directly disintegrated by activated calpain. The in vivo relevance of the findings was tested: pharmacologic inhibition of initial calpain activation augmented early apoptosis of medial VSMCs 24 h after balloon injury in a p53-dependent manner but resulted in a marked increase in late neointima formation. We conclude that calpain counteracts mechanically induced excessive VSMC apoptosis through its p53-degrading properties, which identifies calpain as a key regulator of mechanosensitive remodeling processes of the vascular wall.

Treatment of chronic CAD--do the guidelines (ESC, AHA) reflect daily practice?

In Western countries, chronic coronary artery disease (CAD) has a prevalence of 3-4%. The aims of treatment of chronic CAD are (1) improvement of quality of life by preventing anginal pain, by maintaining exercise capability, and by reducing anxiety; (2) decrease of cardiovascular morbidity, especially by avoiding myocardial infarction and development of heart failure; (3) reduction of mortality. These goals can be achieved by (a) cardiovascular risk reduction, especially management of risk factors, (b) optimal medical therapy, (c) coronary revascularization, (d) periods of rehabilitation, and (e) outpatient long-term observation and treatment. The patient has a good chance to improve the natural course of his disease by changing his lifestyle. In this regard, physical exercise, weight reduction and smoking cessation have to be mentioned first. Furthermore, the cardiovascular risk may significantly be diminished by adequate treatment of hyperlipoproteinemia: lowering of plasma LDL cholesterol levels in patients with chronic CAD is associated with a retarded progression of atherosclerosis as well as a decrease of cardiovascular events by 30-40% and lower mortality (by up to 34%). In patients with CAD and/or type 2 diabetes, statin therapy leads to a significant improvement of prognosis independent of the basal value of LDL cholesterol. Improved diet and adequate medical therapy may also result in diminished cardiovascular risk. By means of physical activity, mortality and morbidity of CAD can also be significantly reduced. The antianginal medication in patients with chronic CAD consists of nitrates, beta-blockers, and calcium channel blockers. In order to prevent myocardial infarction and death (secondary prevention), antiplatelet agents, renin-angiotensin-aldosterone system blockers, as well as cholesterol-lowering drugs are applied. In this paper, the guidelines of the American College of Cardiology/American Heart Association, the European Society of Cardiology and the NVL-KHK (German) guidelines regarding prevention, medical therapy and coronary artery revascularization procedures are summarized. Do the guidelines reflect daily practice? To answer this question, the following topics are discussed: (1) Management of risk factors with respect to available guidelines, (2) missing evidence from randomized controlled trials for medical therapy options widely used in clinical practice, (3) guideline-compliant use or underuse of diagnostic assessment, medical therapy and revascularization procedures, (4) gender bias in indications for percutaneous coronary interventions and in the use of investigations/evidence-based medical therapy, and (5) nonadherence to existing guidelines.

AP-1 and STAT-1 decoy oligodeoxynucleotides attenuate transplant vasculopathy in rat cardiac allografts.

AIMS: Cardiac allograft vasculopathy (CAV) continues to be an unsolved clinical problem requiring the development of new therapeutic strategies. We have previously demonstrated that ex vivo donor allograft treatment with decoy oligodeoxynucleotides (ODN) targeting the transcription factors, activator protein-1 (AP-1) or signal transducer and activator of transcription-1 (STAT-1), delays acute rejection and prolongs cardiac allograft survival. Here, we investigated whether this treatment regime also prevents the occurrence of CAV in a fully allogeneic rat heart transplantation model. METHODS AND RESULTS: Wistar-Furth rat cardiac allografts were perfused ex vivo with AP-1 decoy ODN, STAT-1 decoy ODN, or buffer solution and transplanted into the abdomen of Lewis rats immunosuppressed with cyclosporine. Treatment with both decoy ODNs but not vehicle significantly attenuated the incidence and severity of CAV. Laser-assisted microdissection/real-time polymerase chain reaction as well as immunohistochemistry analyses revealed a significant increase in CD40 abundance in the coronary endothelial cells and medial smooth muscle cells on day 1 post transplantation which was virtually abolished upon AP-1 or STAT-1 decoy ODN treatment. While the AP-1 decoy ODN primarily attenuated basal CD40 expression, the STAT-1 decoy ODN suppressed tumour necrosis factor-alpha-/interferon-gamma-stimulated expression of CD40 in rat native endothelial cells. CONCLUSION: Treating donor hearts with decoy ODNs neutralizing AP-1 or STAT-1 at the time of transplantation prevents upregulation of CD40 expression in the graft coronary arteries and effectively inhibits CAV.

A review of methods for assessment of coronary microvascular disease in both clinical and experimental settings.

Obstructive disease of the large coronary arteries is the prominent cause for angina pectoris. However, angina may also occur in the absence of significant coronary atherosclerosis or coronary artery spasm, especially in women. Myocardial ischaemia in these patients is often associated with abnormalities of the coronary microcirculation and may thus represent a manifestation of coronary microvascular disease (CMD). Elucidation of the role of the microvasculature in the genesis of myocardial ischaemia and cardiac damage-in the presence or absence of obstructive coronary atherosclerosis-will certainly result in more rational diagnostic and therapeutic interventions for patients with ischaemic heart disease. Specifically targeted research based on improved assessment modalities is needed to improve the diagnosis of CMD and to translate current molecular, cellular, and physiological knowledge into new therapeutic options.

Aortic valve stenosis due to alkaptonuria.

Cardiovascular disease is a less-well appreciated aspect of alkaptonuria. A 69-year-old man presented with shortness of breath and exertional chest pain. He had a previous diagnosis of alkaptonuria (endogenous ochronosis), confirmed on the basis of urine coloration, skin pigmentation and ochronotic arthropathy in the knees. Echocardiography and coronary angiography revealed severe aortic valve stenosis and concomitant coronary artery disease. The patient underwent biological aortic valve replacement (AVR) and coronary artery bypass grafting (CABG). Operative findings included ochronosis of a severely calcified aortic valve and the aortic intima, and bioprosthetic AVR and CABG were successfully performed.

Rapamycin attenuates hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy in mice.

BACKGROUND: Chronic hypoxia induces pulmonary arterial hypertension (PAH). Smooth muscle cell (SMC) proliferation and hypertrophy are important contributors to the remodeling that occurs in chronic hypoxic pulmonary vasculature. We hypothesized that rapamycin (RAPA), a potent cell cycle inhibitor, prevents pulmonary hypertension in chronic hypoxic mice. METHODS: Mice were held either at normoxia (N; 21% O2) or at hypobaric hypoxia (H; 0.5 atm; ~10% O2). RAPA-treated animals (3 mg/kg*d, i.p.) were compared to animals injected with vehicle alone. Proliferative activity within the pulmonary arteries was quantified by staining for Ki67 (positive nuclei/vessel) and media area was quantified by computer-aided planimetry after immune-labeling for alpha-smooth muscle actin (pixel/vessel). The ratio of right ventricle to left ventricle plus septum (RV/[LV+S]) was used to determine right ventricular hypertrophy. RESULTS: Proliferative activity increased by 34% at day 4 in mice held under H (median: 0.38) compared to N (median: 0.28, p = 0.028) which was completely blocked by RAPA (median HO+RAPA: 0.23, p = 0.003). H-induced proliferation had leveled off within 3 weeks. At this time point media area had, however, increased by 53% from 91 (N) to 139 (H, p < 0.001) which was prevented by RAPA (H+RAPA: 102; p < 0.001). RV/[LV+S] ratio which had risen from 0.17 (N) to 0.26 (H, p < 0.001) was attenuated in the H+RAPA group (0.22, p = 0.041). For a therapeutic approach animals were exposed to H for 21 days followed by 21 days in H +/- RAPA. Forty two days of H resulted in a media area of 129 (N: 83) which was significantly attenuated in RAPA-treated mice (H+RAPA: 92). RV/[LV+S] ratios supported prevention of PH (N 0.13; H 0.27; H+RAPA 0.17). RAPA treatment of N mice did not influence any parameter examined. CONCLUSION: Therapy with rapamycin may represent a new strategy for the treatment of pulmonary hypertension.

Caveolin-1 facilitates mechanosensitive protein kinase B (Akt) signaling in vitro and in vivo.

Mechanotransduction represents an integral part of vascular homeostasis and contributes to vascular lesion formation. Previously, we demonstrated a mechanosensitive activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) resulting in p27Kip1 transcriptional downregulation and cell cycle entry of vascular smooth muscle cells (VSMC). In this study, we further elucidated the signaling from outside-in toward PI3-K/Akt in vitro and in an in vivo model of elevated tensile force. When VSMC were subjected to cyclic stretch (0.5 Hz at 125% resting length), PI3-K, Akt, and Src kinases were found activated. Disrupting caveolar structures with beta-cyclodextrin or transfection of VSMC with caveolin-1 antisense oligonucleotides (ODN) prevented PI3-K and Akt activation and cell cycle entry. Furthermore, PI3-K and Akt were resistant to activation when Src kinases were inhibited pharmacologically or by overexpression of a kinase-dead c-Src mutant. alpha(V)beta3 integrins were identified to colocalize with PI3-K/caveolin-1 complexes, and blockade of alpha(V)beta3 integrins prevented Akt activation. The central role of caveolin-1 in mechanotransduction was further examined in an in vivo model of elevated tensile force. Interposition of wild-type (WT) jugular veins into WT carotid arteries resulted in a rapid Akt activation within the veins that was almost abolished when veins of caveolin-1 knockout (KO) mice were used. Furthermore, late neointima formation within the KO veins was significantly reduced. Our study provides evidence that PI3-K/Akt is critically involved in mechanotransduction of VSMC in vitro and within the vasculature in vivo. Furthermore, caveolin-1 is essential for the integrin-mediated activation of PI3-K/Akt.

Comparison between pulsed and continuous radiofrequency delivery.

INTRODUCTION: Potentials arising in the pulmonary veins (PV) have been proposed to be a trigger of atrial fibrillation. Percutaneously, the best results for curative treatment of atrial fibrillation have been achieved by segmental or circumferential isolation of the PV. The purpose of our study was to determine the feasibility of ostial pulmonary vein isolation and to compare continuous radiofrequency (RF) with pulsed RF concerning homogeneity and transmurality of produced lesions. MATERIALS AND METHODS: In vivo tests were performed in seven anesthetized and ventilated pigs. Under fluoroscopy and guided by intracardiac electrograms each of the 28 pulmonary veins was targeted for circumferential isolation near its ostium. After the continuous energy application in one PV-ostium the catheter was placed into the next PV-ostium and the same procedure was repeated using pulsed energy. The ablations were performed with an octapolar circumferential ablation catheter, with either continuous RF energy delivery to each electrode for 120 s or pulsed energy delivery to four electrodes simultaneously with a 5 ms duty cycle. Lesion diameter was measured with a microcaliper and homogeneity classified from 1 (highest) to 4 (least). RESULTS: More homogeneous lesions were produced in significantly less time with pulsed rather than with continuous energy delivery. There were no significant differences in impedance or temperature of the electrodes. We did not observe tissue carbonization or "popping," pulmonary vein stenosis, pericardial effusion/perforation at any time. CONCLUSION: Ostial ablation of the PV with pulsed energy delivery proved feasible. It was the faster and more reliable method of creating linear circumferential lesions with a maximum amount of homogeneity and transmurality. We observed no elevated risk of PV stenosis during our experiments.

STAT-1 and AP-1 decoy oligonucleotide therapy delays acute rejection and prolongs cardiac allograft survival.

OBJECTIVE: Acute myocardial rejection is a cell-mediated process characterized by increased leukocyte recruitment into the graft myocardial tissue. Transcription factors like STAT-1 and AP-1 are critically involved in this process by regulating vascular adhesion molecule expression. The aim of our study was to investigate the effect of decoy oligodeoxynucleotide (dODN) treatment targeting transcription factors AP-1 and STAT-1 on acute cardiac allograft rejection in a rat transplant model. METHODS: Wistar-Furth (WF) cardiac allografts were transplanted into Lewis (LEW) rats after perfusion with STAT-1 or AP-1 dODN solution (5 micromol/l), buffer or the corresponding mutant control ODNs. Grafts were harvested and processed for histologic and immunohistochemical evaluation. RESULTS: As demonstrated by fluorescence dye-labelled dODN, exposure of the grafts to the dODNs during 45 min of warm ischemia resulted in a dominant uptake of naked DNA by the graft endothelium. Treatment with AP-1 and STAT-1 dODNs, but not with vehicle or the control dODNs, significantly prolonged cardiac allograft survival by approximately 40% from 5.6+/-0.5 days to 7.8+/-1.3 days and 7.4+/-0.5 days, respectively (mean+/-S.D., p<0.01, n=5 in each group). Immunohistochemical examination on days 1, 3 and 6 revealed a marked reduction of infiltrating leukocytes (AP-1 dODN: 85%, STAT-1 dODN: 50%), namely T-cells, in the dODN-perfused grafts at day 3 post transplantation. In addition, as demonstrated by immunohistochemical analysis, endothelial expression of ICAM-1 and VCAM-1 was found to be markedly reduced in dODN-treated grafts. CONCLUSION: Both AP-1 and STAT-1 dODN treatments suppress graft endothelial adhesion molecule expression, reduce graft infiltration and in turn significantly delay acute rejection. The utilization of dODNs in the cardioplegic solution might be a novel strategy to protect transplanted organs from early damage during transplantation, to preserve organ function and bridge the critical phase after transplantation when standard immunosuppression is not yet completely effective.

Lipopolysaccharide-induced proliferation and adhesion of U937 cells to endothelial cells involves barium chloride sensitive hyperpolarization.

The adhesion of monocytes to the endothelium and their proliferation in the subendothelial space play an important role in atherosclerosis. Since the proliferation and migration of cells are influenced by the activity of ion channels, the aim of this study was to examine whether barium chloride (Ba(2+))-sensitive potassium channels (K(iCa)) are involved in lipopolysaccharide (LPS)-induced proliferation of monocytic U937 cells, and in the adhesion of these cells to endothelial cells. The adhesion of LPS-stimulated U937 cells to endothelial cells reached a maximum at a concentration of 5 microg/ml. This effect of LPS was completely abolished in the presence of Ba(2+) (100 micromol/l). In addition, LPS-induced proliferation was significantly reduced by Ba(2+) (control, 100%; LPS 5 microg/ml, 175%; LPS + Ba(2+) 100 micromol/l, 136%; n = 12, P < 0.05). To examine whether K(iCa) are activated by LPS, changes of U937 membrane potential were determined. LPS (5 microg/ml) caused a hyperpolarization of U937 cells indicating a flux of K(+) ions out of the cells. This effect was completely blocked by Ba(2+) (100 micromol/l). In conclusion, we demonstrate that LPS activates K(iCa) in U937 cells, which is responsible for LPS-induced adhesion of these cells to endothelial cells, and to the proliferation of U937 cells.