ß-blockade abolishes the augmented cardiac tPA release induced by transactivation of heterodimerised bradykinin receptor-2 and ß2-adrenergic receptor in vivo.

Bradykinin (BK) receptor-2 (B2R) and ß2-adrenergic receptor (ß2AR) have been shown to form heterodimers in vitro. However, in vivo proofs of the functional effects of B2R-ß2AR heterodimerisation are missing. Both BK and adrenergic stimulation are known inducers of tPA release. Our goal was to demonstrate the existence of B2R-ß2AR heterodimerisation in myocardium and to define its functional effect on cardiac release of tPA in vivo. We further investigated the effects of a non-selective ß-blocker on this receptor interplay. To investigate functional effects of B2R-ß2AR heterodimerisation (i. e. BK transactivation of ß2AR) in vivo, we induced serial electrical stimulation of cardiac sympathetic nerves (SS) in normal pigs that underwent concomitant BK infusion. Both SS and BK alone induced increases in cardiac tPA release. Importantly, despite B2R desensitisation, simultaneous BK infusion and SS (BK+SS) was characterised by 2.3 ± 0.3-fold enhanced tPA release compared to SS alone. When ß-blockade (propranolol) was introduced prior to BK+SS, tPA release was inhibited. A persistent B2R-ß2AR heterodimer was confirmed in BK-stimulated and non-stimulated left ventricular myocardium by immunoprecipitation studies and under non-reducing gel conditions. All together, these results strongly suggest BK transactivation of ß2AR leading to enhanced ß2AR-mediated release of tPA. Importantly, non-selective ß-blockade inhibits both SS-induced release of tPA and the functional effects of B2R-ß2AR heterodimerisation in vivo, which may have important clinical implications.

Different cardiac tissue plasminogen activator release patterns by local stimulation of the endothelium and sympathetic nerves in pigs.

Myocardial ischemia induces cardiac tissue plasminogen activator (tPA) release, declining by repeated periods of ischemia. However, the mechanisms and cellular sources are unknown. Sympathetic nerve stimulation (SS) and bradykinin (BK), an endogenous inducer of endothelial tPA release, may play roles, potentially involving different sources or mechanisms revealed by different release patterns. Therefore, we compared the cardiac tPA release patterns during repeated coronary BK infusions and SS, both with an ensuing period of local myocardial ischemia/reperfusion (I/R). Nine pigs were subjected to four periods of coronary BK infusion (4 min) and another nine animals to four periods of SS (4 min). Finally, 10 min of I/R was induced in both groups. The single-peaked BK-induced tPA release declined toward baseline by repeated infusions, but tPA release reappeared during I/R. In contrast, total tPA release during repeated SS and subsequent I/R was more stable, and SS-induced total tPA and norepinephrine (NE) releases were strongly correlated. Surprisingly, the instantaneous SS-induced tPA release was biphasic with a stable first peak, and a second peak declining toward baseline by repeated stimulations. The fluctuations in cardiac release of plasminogen activator inhibitor-1 and the endogenous BK inhibitor angiotensin-converting enzyme, could not explain the diverging tPA release patterns. Different tPA release patterns were demonstrated during SS and BK stimulation, as well as diverging responses to repeated stimulations and subsequent I/R. This study demonstrates strong association between tPA and NE during SS and possibly two different sources or mechanisms for SS-induced tPA release.

Circulating CD34⁺ progenitor cells and growth factors in patients treated with PCI for acute myocardial infarction or stable angina pectoris.

OBJECTIVE: To differentiate the effect of myocardial infarction from the effect of percutaneous coronary intervention (PCI) on the circulatory profiles of CD34(+) cells and growth factors in patients with ST-elevation myocardial infarction (STEMI). METHODS: Twenty patients with STEMI and 10 with angina pectoris (AP) were included. All were treated with PCI. Blood was drawn before PCI in the AP group, and after 3 and 12 hours, and 1, 3, 5, 7 and 14 days after PCI in both groups. In STEMI patients, correlation analyses between TIMI myocardial perfusion grade (TMP-grade) and circulating CD34(+) cells were also assessed. RESULTS: Circulating CD34(+) cells increased from day 1 to days 5 and 7 after PCI only in STEMI patients (p < 0.05). Between-group analyses revealed a borderline significant difference in change in SDF-1α concentrations from 3 h to 14 days after PCI (p = 0.05), and SDF-1α was significantly higher in STEMI patients 14 days after PCI (p < 0.05). In both groups, peak HGF concentrations were observed 3 h after PCI, whereas IGF-1 increased in AP patients only, 3 h after PCI (p < 0.005). TIMI perfusion grade was negatively correlated to the circulating number of CD34(+) cells 5 days after PCI (r =-0.69, p < 0.005). CONCLUSION: After PCI, STEMI patients have significantly higher numbers of circulating CD34(+) progenitor cells compared to patients with AP. STEMI results in a significant increase in SDF-1α after 14 days, and the increase at this time may indicate a favorable environment for progenitor cell therapy.

Cell treatment after acute myocardial infarction prevents early decline in circulating IGF-1.

OBJECTIVES: To examine the influence of intracoronary autologous bone marrow cell transplantation after acute myocardial infarction on circulating growth factors and their relationship to left ventricular function. METHODS: Circulating insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), stromal derived factor-1-alpha (SDF-1α), and transforming growth factor beta (TGF-ß) were measured in patients randomized to cell treatment or control, in the ASTAMI study. Autologous cells were injected intracoronary on day 6; blood was sampled on days 5, 9, and at three months. Left ventricular ejection fraction was recorded by electrocardiogram-gated single photon emission computed tomography at six months. RESULTS: Only change in IGF-1 from baseline to three months differed between groups (p = 0.024). A weak but significant correlation was found between left ventricular ejection fraction and the averaged IGF-1 concentrations of all patients (r = 0.24, p = 0.02). Patients with IGF-1 above or below median (102 ng/ml) had a left ventricular ejection fraction of 52.3% (±11.4) versus 46.4% (±12.2) respectively (p = 0.017). CONCLUSIONS: Intracoronary bone marrow cell treatment after myocardial infarction attenuates a reduction in circulating IGF-1. IGF-1 levels over time were weakly, but significantly correlated to left ventricular ejection fraction.

Release of tissue-type plasminogen activator (t-PA) in the splanchnic circulation of the anaesthetised pig during high sympathetic tone.

BACKGROUND: There is a substantial local release of tissue-type plasminogen activator (t-PA) in the splanchnic vascular bed, and this release is increased at high sympathetic tone. Coronary t-PA release is also significant, and this release increases during cardiac nerve stimulation and during reperfusion after 10 min of local myocardial ischemia. However, by repeated cycles of myocardial ischemia/reperfusion coronary t-PA release progressively declines. OBJECTIVES: Accordingly, we hypothesised that splanchnic t-PA release might decrease after an initial peak during maintained and long-lasting sympathetic stimulation. METHODS: In 6 anaesthetised pigs sympathetic tone was augmented by bleeding (20-25 mL/kg over 30 minutes). During the subsequent 2 hours period portal vein (draining the splanchnic vascular bed) - and arterial blood samples were drawn every 20 min and portal vein blood flow was recorded continuously in order to estimate t-PA release in the splanchnic vascular bed. RESULTS: Relatively stable haemodynamic conditions were obtained after bleeding with mean arterial blood pressure at 50 to 65 mmHg and a portal vein flow at about the 50% of baseline value. Net splanchnic t-PA release rose to a peak 40 min after bleeding, but subsequently declined towards baseline values. Arterial t-PA activity rose after the bleeding period and to a peak value at end of the observation period. CONCLUSIONS: Net splanchnic t-PA release increased only transiently during the period with increased sympathetic stimulation, whereas the arterial t-PA level remained elevated. During a strong and longlasting sympathetic stimulation the lack of a continuously augmented splanchnic t-PA release might increase the risk for intravenous splanchnic thrombosis.

Exercise capacity and quality of life after intracoronary injection of autologous mononuclear bone marrow cells in acute myocardial infarction: results from the Autologous Stem cell Transplantation in Acute Myocardial Infarction (ASTAMI) randomized controlled trial.

BACKGROUND: The effects on left ventricular function of intracoronary injection of bone marrow cells in acute myocardial infarction (AMI) have been studied with conflicting results. The aim of this substudy of the ASTAMI trial was to examine the effects of this novel treatment on exercise capacity and quality of life. METHODS: We studied 100 patients with anterior wall ST-elevation AMI. All had percutaneous coronary intervention with stent in the proximal or mid left anterior descending coronary artery 2 to 12 hours after start of symptoms. Patients were randomized to intracoronary injection of mononuclear bone marrow cells (mBMCs) in left anterior descending coronary artery 6 +/- 1.3 days after AMI (n = 50) or control (n = 50). Assessment of physical capacity by maximal symptom-limited bicycle ergometer exercise tests and quality of life by the Short Form 36 health survey was performed 2 to 3 weeks and 6 months after the AMI. RESULTS: There was a significantly greater improvement in exercise time in the mBMC group than in the control group (treatment effect 0.9 minute, 95% CI 0.3-1.6, P < .01), and a similar improvement in peak oxygen consumption in the groups (2.8 +/- 3.9 mL/[kg min] in the mBMC group vs 2.4 +/- 3.5 mL/[kg min] in controls, P = .62). Peak heart rate and percentage of heart rate reserve increased significantly more in the treatment group than in the control group. Treatment with mBMCs did not influence quality of life. CONCLUSIONS: In this randomized open-labeled study, the mBMC group significantly improved exercise time and heart rate responses to exercise compared with the control group. There was no treatment effect on peak oxygen consumption.

Cardiac accumulation of bone marrow mononuclear progenitor cells after intracoronary or intravenous injection in pigs subjected to acute myocardial infarction with subsequent reperfusion.

OBJECTIVE: The purpose of the present study was to compare the efficacy of intracoronary and intravenous injection of autologous progenitor cells for homing to the acutely infarcted but reperfused myocardium in pigs. METHODS: Myocardial infarction was induced in 11 anesthetized pigs by 60-min balloon inflation in the mid LAD. After balloon deflation, reperfusion was verified and autologous CD31(+) progenitor cells, or bone marrow mononuclear cells, labeled with PKH67, were injected either intracoronarily (n=6) or intravenously (n=3). By autopsy, 4-5 days after induction of infarction, tissue from the heart and other organs was obtained for fluorescence microscopy. RESULTS: In the heart, PKH(+) cells were detected throughout the reperfused infarcted myocardium, and the number of PKH(+) cells was significantly higher after intracoronary than after intravenous injection (3.2+/-0.55 vs. 0.33+/-0.17 cells/high-power field/10(6) cells injected, P=.01). Few PKH(+) cells were detected in the spleen, lung, mesenteric lymph node, and bone marrow. In an additional animal with a coil placed in the mid LAD, progenitor cells were not detected in the infarcted myocardium or in the normal myocardium. CONCLUSION: Autologous mononuclear and CD31(+) cells from bone marrow accumulated in the infarcted myocardium when injected intracoronarily or intravenously after established reperfusion, and the accumulation of cells was significantly greater after intracoronary injection than after intravenous injection. Accumulation of PKH(+) cells did not appear in the normal myocardium or in the nonreperfused infarcted myocardium. PKH(+) cells were detected in spleen, lung, and bone marrow but to a lesser degree than in the infarcted myocardium.

Ventricular fibrillation induced by ischemia-reperfusion is not prevented by the NPY Y2 receptor antagonist BIIE0246.

Neuropeptide Y is released together with norepinephrine from sympathetic nerve terminals during conditions of increased sympathetic activity. Neuropeptide Y is known to inhibit vagal activity, and accordingly, it might increase the risk for ventricular fibrillation during myocardial ischemia-reperfusion, with concomitant sympathetic stimulation. Counteracting the inhibiting effect of neuropeptide Y by the specific neuropeptide Y2 antagonist, BIIE0246, we expected occurrence of ventricular fibrillation in association with repeated periods of myocardial ischemia-reperfusion to decrease. The midleft anterior descending coronary artery was repeatedly occluded in 16 open-chest pigs. Eight pigs received BIIE0246, and the controls received the vehicle only. Ventricular fibrillation developed in 2 animals of the control group, but in 4 pigs receiving BIIE0246. Occurrence of ventricular fibrillation and ventricular tachycardia did not differ significantly between the 2 groups, and in association with repeated periods of regional myocardial ischemia, did not decline in pigs treated by the specific neuropeptide Y2-receptor antagonist BIIE0246.

Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction.

BACKGROUND: Previous studies have shown improvement in left ventricular function after intracoronary injection of autologous cells derived from bone marrow (BMC) in the acute phase of myocardial infarction. We designed a randomized, controlled trial to further investigate the effects of this treatment. METHODS: Patients with acute ST-elevation myocardial infarction of the anterior wall treated with percutaneous coronary intervention were randomly assigned to the group that underwent intracoronary injection of autologous mononuclear BMC or to the control group, in which neither aspiration nor sham injection was performed. Left ventricular function was assessed with the use of electrocardiogram-gated single-photon-emission computed tomography (SPECT) and echocardiography at baseline and magnetic resonance imaging (MRI) 2 to 3 weeks after the infarction. These procedures were repeated 6 months after the infarction. End points were changes in the left ventricular ejection fraction (LVEF), end-diastolic volume, and infarct size. RESULTS: Of the 50 patients assigned to treatment with mononuclear BMC, 47 underwent intracoronary injection of the cells at a median of 6 days after myocardial infarction. There were 50 patients in the control group. The mean (+/-SD) change in LVEF, measured with the use of SPECT, between baseline and 6 months after infarction for all patients was 7.6+/-10.4 percentage points. The effect of BMC treatment on the change in LVEF was an increase of 0.6 percentage point (95% confidence interval [CI], -3.4 to 4.6; P=0.77) on SPECT, an increase of 0.6 percentage point (95% CI, -2.6 to 3.8; P=0.70) on echocardiography, and a decrease of 3.0 percentage points (95% CI, 0.1 to -6.1; P=0.054) on MRI. The two groups did not differ significantly in changes in left ventricular end-diastolic volume or infarct size and had similar rates of adverse events. CONCLUSIONS: With the methods used, we found no effects of intracoronary injection of autologous mononuclear BMC on global left ventricular function.

Influence of endogenous neuropeptide Y (NPY) on the sympathetic-parasympathetic interaction in the canine heart.

The purpose of this study was to examine the sympathetic-parasympathetic interactions on heart rate through release of neuropeptide Y (NPY) and its action on prejunctional NPY Y2 receptors on vagal and sympathetic nerve fibers. In other studies on various preparations and in various organs, attenuation of transmitter release has in fact been demonstrated through activation of the NPY Y2 receptor. In the present study on anesthetized dogs we examine, however, for the first time if vagal bradycardia is attenuated by endogenous NPY released during intense cardiac sympathetic stimulation. In addition, we explore if sympathetic transmitter release and heart rate, during moderate sympathetic stimulation, are affected through this receptor system. The significance of the NPY Y2 receptor was revealed by performing experiments before and after administration of its specific receptor antagonist BIIE0246. We found that attenuation of the bradycardia during vagal nerve stimulation was dose-dependently counteracted by BIIE0246 and that the tachycardia elicited by sympathetic stimulation remained unaffected after NPY Y2 receptor blockade. Thus, endogenous NPY appears to attenuate vagal bradycardia by stimulating prejunctional NPY Y2 receptors on cardiac vagal nerve terminals and, less efficiently, to attenuate transmitter release and tachycardia through a feedback loop on the cardiac sympathetic nerve fibers.

Reduced release of vasoconstrictors from the porcine heart after repeated periods of ischemia.

OBJECTIVE: To examine if the decline in post-ischemic hyperemic flow after repeated brief periods of myocardial ischemia is accompanied by augmented cardiac release of the vasoconstrictors endothelin-1 (ET-1) and norepinephrine (NE). DESIGN: Mid-LAD (left anterior descending coronary artery) was occluded for 10 min with 30 min intervals a total of four times in six anesthetized pigs. Blood from the anterior interventricular coronary vein was drained through a shunt to the right atrium to facilitate blood sampling. Plasma concentrations of ET-1 and NE were repeatedly measured in arterial and coronary venous blood to estimate cardiac vasoconstrictor release. RESULTS: Plasma concentrations of ET-1 and NE remained unaltered, but cardiac release of both vasoconstrictors rose briefly during reperfusion due to the hyperemia. However, release declined progressively after repeated periods of ischemia and reperfusion and amounted to 53% (NE) and 17% (ET-1) of initial release after the fourth period of ischemia. CONCLUSION: The decline in post-ischemic hyperemia after repeated brief periods of myocardial ischemia is not accompanied by a progressive accentuation of cardiac ET-1 and NE release.

Evaluation of a new fiber-optic pressure recording system for cardiovascular measurements in mice.

We have tested a new fiber-optic pressure recording system, Samba, with a thin fiber [outer diameter (OD) = 0.25 mm] and a pressure sensor (length and OD = 0.42 mm) attached to the end. The accuracy of the system tested in vitro was good, with a coefficient of variation of 2.54% at 100 mmHg. The drift was <0.45 mmHg/h, and the temperature sensitivity was approximately 0.07 mmHg/1 degrees C between 22 and 37 degrees C. The frequency response characteristics were similar to a 1.4-Fr Millar catheter (0-200 Hz). Introduction of the Samba sensor from the right carotid artery into the left ventricle in six mice caused no drop in mean aortic pressure, whereas introduction of a 1.4-Fr Millar catheter (OD = 0.47 mm; n = 6) caused a pressure drop from 91.6 +/- 9.2 to 65.1 +/- 6.2 mmHg; P < 0.05. Thus the Samba sensor system may represent a new alternative to assess hemodynamic variables in the murine cardiovascular system.

Distribution of neuropeptide Y Y1 and Y2 receptors in the postmortem human heart.

In the present study, we present for the first time the presence and distribution of neuropeptide Y (NPY) receptors Y1 and Y2 in the human postmortem heart using specific antibodies raised against extracellular parts of the receptors. A more intensive staining against the Y2 than against the Y1 receptors was detected on both atrial and ventricular cardiomyocytes. Immunoreactivity against both receptors was identified on both conducting fibers and cardiac nerves. More vessels stained positively for the Y2 than for the Y1 receptor, but the Y1 receptors were more abundant in subendocardial than subepicardial vessels of the left ventricular wall.