Ruth H. Strasser

Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel® under angiogenic conditions

OBJECTIVES It has been shown that circulating human non-adherent CD34+ cells coexpressing vascular endothelial growth factor (VEGF)-R2 and AC133 have the capacity to differentiate into adherent mature endothelial cells. However, prior studies have demonstrated that a much bigger subset of primary adherent mononuclear cells can also form endothelial progenitor cells (EPC). To determine the origin of the latter cell population we tested the hypothesis: do monocytes as a firmly adherent and plastic cell type have the potential to differentiate into an endothelial phenotype. METHODS CD34-/CD14+ monocytes were isolated from human peripheral blood by adherence separation and magnetic bead selection (purity >90%) and cultured on fibronectin-coated plastic dishes (medium containing VEGF 10 ng/ml, basic fibroblast growth factor (bFGF) 2 ng/ml, insulin like growth factor (IGF-1) 1 ng/ml, 20% fetal calf serum). RESULTS After 2 weeks of culture, using fluorescence activated cell analysis we observed a new expression of the endothelial markers von Willebrand factor (vWf), VE-cadherin (VE) and ec-NOS in 45.2, 12.4 and 9.8% of the cells, respectively. The proportion of cells expressing these markers further increased after 4 weeks (94.2, 89.7 and 58.8% of these cells, respectively). The proportion of CD45 expressing cells remained unchanged during this period. However, after 14 days the specific macrophage antigen CD68 was newly expressed in 62% of the analysed cells with a further increase to 90% after 28 days of culture. In three-dimensional gel (Matrigel the formation of cord- and tubular-like structures was observed. CONCLUSION The present data indicate that under angiogenic stimulation macrophages develop an endothelial phenotype with expression of specific surface markers and even form cord- and tubular-like structures in vitro suggesting that this cell population may be recruited for vasculogenesis.

Transcatheter closure of patent foramen ovale in patients with cerebral ischemia

OBJECTIVES The present study was conducted to determine the safety of the transcatheter closure of a patent foramen ovale (PFO) in patients with cryptogenic cerebral ischemia and the midterm follow-up of recurrent thromboembolic events after interventional PFO closure. BACKGROUND Current therapeutic options for stroke prevention in patients with PFO and a history of thromboembolic events include chronic antithrombotics and more invasive treatments such as surgical closure or minor invasive transcatheter permanent closure of the PFO. Promising preliminary and pilot data with the Amplatzer Septal Occluder or the PFO-Star Occluder have been reported. Systematic and long-term data are still missing. METHODS A total of 276 consecutive patients with a PFO and a history of at least one thromboembolic event were recruited in four medical centers and underwent percutaneous PFO closure with the PFO-Star device. Follow-up data were analyzed over an average of 15.1 months, equivalent to 345 patient-years. RESULTS The implantation was successful in all 276 patients. Peri-interventional reversible complications included transient ST-segment elevations (1.8%) and transient ischemic attack (TIA) (0.8%). Two devices have been removed surgically. During follow-up the annual recurrence rate of thromboembolic events was 1.7% for TIA, 0% for stroke and 0% for peripheral emboli. CONCLUSIONS Interventional PFO closure with the PFO-Star device appears to be a reliable and promising technique resulting in a low recurrence rate of thromboembolic events, especially stroke in patients with a history of cryptogenic ischemia presumably due to paradoxical embolization. To our knowledge, this is the largest coherent and prospective study for interventional PFO closure.

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 &bgr;-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. &agr;V&bgr;3 integrins were identified to colocalize with PI3-K/caveolin-1 complexes, and blockade of &agr;V&bgr;3 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.

Alpha 1-receptor-independent activation of protein kinase C in acute myocardial ischemia. Mechanisms for sensitization of the adenylyl cyclase system.

The activity of the adrenergic system plays an important role in the genesis of malignant arrhythmias and the spreading of the infarcted zone in acute myocardial ischemia. Acute myocardial ischemia induces an increased activity of adenylyl cyclase. This sensitization at the enzyme level as shown in the isolated perfused rat heart occurs rapidly after the onset of ischemia (5-15 minutes) and is rapidly reversible on reperfusion. With prolonged ischemia, it is only transient and is followed by a gradual loss of the adenylyl cyclase activity. The increased activity of adenylyl cyclase is even retained after partial purification, suggesting a covalent modification of the enzyme. Blockade of alpha 1-adrenergic receptors does not prevent this sensitization, demonstrating that it occurs independently of alpha 1-adrenergic receptor activation. Only blockade of protein kinase C by various inhibitors, such as polymyxin B or staurosporine, is able to completely prevent this sensitization process. Moreover, in acute myocardial ischemia an activation of protein kinase C could be identified using its translocation from the cytosol to the particulate fraction as an indicator. Blockade of alpha 1-adrenergic receptors using prazosin fails to prevent the activation of protein kinase C and consequently the sensitization of the adenylyl cyclase system, indicating that the ischemia-induced translocation of protein kinase C occurs independently of alpha 1-adrenergic receptors. These data characterize for the first time an important interaction of two effector enzymes of two distinct signal transduction pathways, i.e., the adenylyl cyclase system and the protein kinase C system in acute myocardial ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Remote preconditioning by infrarenal occlusion of the aorta protects the heart from infarction: a newly identified non-neuronal but PKC-dependent pathway

BACKGROUND Ischemic preconditioning is a powerful mechanism in reducing infarct size of the heart. Protection can be performed either by an ischemic stimulus of the heart itself or by ischemia of an organ distant to the heart. To address the question whether this remote preconditioning is transduced by neuronal or humoral factors an in situ model of infrarenal occlusion of the aorta (IOA) in the rat was developed. Furthermore, the signal transduction pathways of classical and remote preconditioning regarding protein kinase C, which is one of the key enzymes in classical preconditioning, were compared. METHODS AND RESULTS Controls (30 min regional ischemia followed by 2 h of reperfusion) had an infarct size of 62+/-5% whereas classical preconditioning reduced it to 10+/-3% of the risk zone (P< or =0.001). Fifteen minutes IOA without reperfusion of the aorta had no influence on infarct size (52+/-4%). When, however, IOA was performed for 15, 10, or 5 min, respectively, followed by a 10-min reperfusion period the size of myocardial infarction decreased significantly. This decrease was dependent on the duration of IOA (18+/-3%, 37+/-8%, 42+/-2%, respectively; P< or =0.001 for the time-dependent linear trend in decrease of infarct size). Fifteen minutes IOA showed the strongest protection which was comparable to classical preconditioning (18+/-3%, P< or =0.001 vs. control). Blockade of the nervous pathway by 20 mg/kg hexamethonium could not inhibit the protection afforded by IOA (14+/-4%). Using chelerythrine, a selective protein kinase C-inhibitor, at a dose of 5 mg/kg body weight, protection from remote (68+/-4%, P< or =0.001 vs. 15 min IOA followed by 10 min of reperfusion without chelerythrine) as well as from classical preconditioning (56+/-5%, P< or =0.001) was completely blocked. CONCLUSION Protection of the heart by remote preconditioning using IOA is as powerful as classical preconditioning. Both protection methods share protein kinase C as a common element in their signal transduction pathways. Since hexamethonium could not block the protection from IOA and a reperfusion period has to be necessarily interspaced between the IOA and the infarct inducing ischemia of the heart, a neuronal signal transmission from the remote area to the heart can be excluded with certainty. A humoral factor must be responsible for the remote protection. Interestingly the production of the protecting factor is dependent on the duration of the ischemia of the lower limb. The protecting substance, which must be upstream of protein kinase C, remains to be identified.

Levosimendan is superior to enoximone in refractory cardiogenic shock complicating acute myocardial infarction.

Objective:Cardiogenic shock is the leading cause of death in patients hospitalized for acute myocardial infarction. The objectives were to investigate the effects of levosimendan, a novel inodilator, compared with the phosphodiesterase-III inhibitor enoximone in refractory cardiogenic shock complicating acute myocardial infarction, on top of current therapy. Design:Prospective, randomized, controlled single-center clinical trial. Setting:Medical and coronary intensive care unit in a university hospital. Patients:Thirty-two patients with refractory cardiogenic shock for at least 2 hrs requiring additional therapy. Interventions:Infusion of either levosimendan (12 &mgr;g/kg over 10 min, followed by 0.1 &mgr;g/kg/min over 50 min, and of 0.2 &mgr;g/kg/min for the next 23 hrs) or enoximone (fractional loading dose of 0.5 mg/kg, followed by 2–10 &mgr;g/kg/min continuously) after initiation of current therapy, always including revascularization, intra-aortic balloon pump counterpulsation, and inotropes. Measurements and main results:Survival rate at 30 days was significantly higher in the levosimendan-treated group (69%, 11 of 16) compared with the enoximone group (37%, 6 of 16, p = 0.023). Invasive hemodynamic parameters during the first 48 hrs were comparable in both groups. Levosimendan induced a trend toward higher cardiac index, cardiac power index, left ventricular stroke work index, and mixed venous oxygen saturation. In addition, lower cumulative values for catecholamines at 72 hrs and for clinical signs of inflammation were seen in the levosimendan-treated patients. Multiple organ failure leading to death occurred exclusively in the enoximone group (4 of 16 patients). Conclusions:In severe and refractory cardiogenic shock complicating acute myocardial infarction, levosimendan, added to current therapy, may contribute to improved survival compared with enoximone.

Inotropic response to β-adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts

1 The functional consequences of a lack of endothelial nitric oxide synthase (eNOS) on left ventricular force development and the anti‐adrenergic effect of acetylcholine (ACh) were investigated in isolated hearts and cardiomyocytes from wild type (WT) and eNOS knockout (eNOS–/–) mice. 2 eNOS expression in cardiac myocytes accounted for 20 % of total cardiac eNOS (Western blot analysis). These results were confirmed by RT‐PCR analysis. 3 In the unstimulated perfused heart, the left ventricular pressure (LVP) and maximal rate of left ventricular force development (dP/dtmax) of eNOS–/– hearts were not significantly different from those of WT hearts (LVP: 97 ± 11 mmHg WT vs. 111 ± 11 mmHg eNOS–/–; dP/dtmax: 3700 ± 712 mmHg s−1 WT vs. 4493 ± 320 mmHg s−1 eNOS–/–). 4 The dobutamine (10‐300 nm)‐induced increase in LVP was enhanced in eNOS–/– hearts. In contrast, L‐type Ca2+ currents (ICa,L) in isolated cardiomyocytes of WT and eNOS–/– hearts showed no differences after β‐adrenergic stimulation. Dibutyryl‐cGMP (50 μm) reduced basal ICa,L in WT cells to 72 ± 12 % while eNOS–/–ICa,L was insensitive to the drug. The pre‐stimulated ICa,L (30 nm isoproterenol) was attenuated by dibutyryl‐cGMP in WT and eNOS–/– cells to the same extent. 5 The Ca2+ (1.5‐4.5 mm)‐induced increase in inotropy was not different between the two experimental groups and β‐adrenergic receptor density was increased by 50 % in eNOS–/– hearts. 6 The contractile effects of dobutamine could be inhibited almost completely by ACh or adenosine. The extent of the anti‐adrenergic effect of both compounds was identical in WT and eNOS–/– hearts. Measurement of ICa,L in isolated cardiac myocytes yielded similar results. 7 These data demonstrate that in the adult mouse (1) lack of eNOS is associated with increased cardiac contractile force in response to β‐adrenergic stimulation and with elevated β‐adrenergic receptor density, (2) the unaltered response of ICa,L in eNOS–/– cardiac myocytes to β‐adrenergic stimulation suggests that endothelium‐derived NO is important in mediating the whole‐organ effects and (3) eNOS is unimportant for the anti‐adrenergic effect of ACh and adenosine.

Intraaortic balloon counterpulsation in acute myocardial infarction complicated by cardiogenic shock: Design and rationale of the Intraaortic Balloon Pump in Cardiogenic Shock II (IABP-SHOCK II) trial

BACKGROUND In current guidelines, intraaortic balloon pumping (IABP) is considered a class 1 indication in cardiogenic shock complicating acute myocardial infarction. However, evidence is mainly based on retrospective or prospective registries with a lack of randomized clinical trials. Therefore, IABP is currently only used in 20% to 40% of cardiogenic shock cases. The hypothesis of this trial is that IABP in addition to early revascularization by either percutaneous coronary intervention or coronary artery bypass grafting will improve clinical outcome of patients in cardiogenic shock. STUDY DESIGN The IABP-SHOCK II study is a 600-patient, prospective, multicenter, randomized, open-label, controlled trial. The study is designed to compare the efficacy and safety of IABP versus optimal medical therapy on the background of early revascularization by either percutaneous coronary intervention or coronary artery bypass grafting. Patients will be randomized in a 1:1 fashion to 1 of the 2 treatments. The primary efficacy end point of IABP-SHOCK II is 30-day all-cause mortality. Secondary outcome measures, such as hemodynamic, laboratory, and clinical parameters, will serve as surrogate end points for prognosis. Furthermore, an intermediate and long-term follow-up at 6 and 12 months will be performed. Safety will be assessed, by the GUSTO bleeding definition, peripheral ischemic complications, sepsis, and stroke. CONCLUSIONS The IABP-SHOCK II trial addresses important questions regarding the efficacy and safety of IABP in addition to early revascularization in patients with cardiogenic shock complicating myocardial infarction.

High rates of prasugrel and ticagrelor non-responder in patients treated with therapeutic hypothermia after cardiac arrest.

INTRODUCTION After cardiac arrest due to acute coronary syndromes (ACS) therapeutic hypothermia (HT) is the standard care to reduce neurologic damage. Additionally, the concomitant medical treatment with aspirin and a P2Y12 receptor inhibitor like clopidogrel (Cl), prasugrel (Pr) or ticagrelor (Ti) is mandatory. The platelet inhibitory effect of these drugs under hypothermia remains unclear. METHODS 164 patients with ACS were prospectively enrolled in this study. 84 patients were treated with HT, 80 patients were under normothermia (NT). All patients were treated with aspirin and one of the P2Y12 receptor inhibitors Cl, Pr or Ti. 24h after the initial loading dose the platelet reactivity index (PRI/VASP-index) was determined to achieve the platelet inhibitory effect. RESULTS In the HT-group the PRI/VASP-index was significantly higher compared to the NT-group (54.86%±25.1 vs. 28.98%±22.8; p<0.001). In patients under HT receiving Cl, the platelet inhibition was most markedly reduced (HT vs. NT: 66.39%±19.1 vs. 33.36%±22.1; p<0.001) compared to Pr (HT vs. NT: 37.6%±25.0 vs. 27.04%±25.5; p=0.143) and Ti (HT vs. NT: 41.5%±21.0 vs. 17.83%±14.5; p=0.009). The rate of non-responder defined as PRI/VASP-index>50% was increased in HT compared to NT (60.7% vs. 22.5%; p<0.001) with the highest rates in the group receiving Cl (CL: 82% vs. 26%, p<0.001; Pr: 32% vs. 23%; n.s.; Ti: 30% vs. 8%, n.s.). CONCLUSION The platelet inhibitory effect in patients treated with HT after cardiac arrest is significantly reduced. This effect was most marked with the use of Cl. The new P2Y12-inhibitors Pr and Ti improved platelet inhibition in HT, but could not completely prevent non-responsiveness.

Dual sensitization of the adrenergic system in early myocardial ischemia: Independent regulation of the β-adrenergic receptors and the adenylyl cyclase

Acute myocardial ischemia provokes sensitization of the adenylyl cyclase system. This sensitization can be differentiated in a receptor-specific and an enzyme-specific sensitization. The receptor-linked sensitization is characterized by an increase of beta-adrenergic receptors in the plasma membranes after 15 mins of global ischemia (49.8 +/- 3.6 to 67 +/- 6 fmol/mg protein) followed by a further increase (89 +/- 4 fmol/mg protein) after 50 min of ischemia in isolated perfused hearts. Concomitantly functionally coupled receptors which are able to bind the beta-agonist with high affinity, increased by 32% after 15 min and by 57% after 50 min of ischemia. The affinities of the receptors for their agonists or their antagonists remain unchanged. Maximally isoproterenol-stimulated adenylyl cyclase activity rose from 66 +/- 7 to 101 +/- 10 pmol cAMP/min/mg protein after 15 min of global ischemia indicating the beta-receptor-specific sensitization of the beta-adrenergic system. This sensitization was followed by a gradual decline of the adenylyl cyclase activity after 30 and 50 min of global ischemia. Additionally, 15 min of myocardial ischemia induced an enzyme-linked sensitization of the adenylyl cyclase activity as indicated by an increase of the forskolin-stimulated activity by about 25% (300 +/- 20 vs 378 +/- 25 pmol cAMP/min/mg protein). In contrast after 50 min of ischemia the total adenylyl cyclase activity declined (232 +/- 24 pmol cAMP/min/mg protein) despite the persistent increase of beta-adrenergic receptors in the plasma membranes. These data demonstrate that the enzyme-specific sensitization is only transient. The early sensitization and late inactivation of the adenylyl cyclase activity occurred independently of receptor activation and could not be prevented by beta-blockade (10(-6) M alprenolol). Cyanide perfusion (1 mM), used to block energy metabolism, lead to energy depletion similar to acute myocardial ischemia. This resulted in an increase of functionally coupled receptors with a time course comparable to that of global ischemia. Additional perfusion with desensitizing concentrations of the beta-agonist isoproterenol did not induce uncoupling or internalization of beta-adrenergic receptors in cyanide treated hearts, suggesting that the rise in functionally coupled receptors is due to a redistribution in part caused by the abolition of continuous receptor internalization. In contrast, the enzyme-linked sensitization is independent of cellular localization of the beta-adrenergic receptors. The increased activity was carried by the enzyme even after partial purification with solubilization and wheat germ affinity chromatography. These data suggest an ischemia-induced, covalent modification of the adenylyl cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)