Johannes Wiecha

Blockade of Ca2+-Activated K+ Channels Inhibits Proliferation of Human Endothelial Cells Induced by Basic Fibroblast Growth Factor

Basic fibroblast growth factor (bFGF) exerts angiogenic and mitogenic properties in human tissue. Since changes in ion currents modulate essential Ca2+-dependent intracellular pathways in endothelial cells, we have investigated a possible contribution of Ca2+-activated K+ channels (BKCa) on bFGF-induced endothelial cell proliferation. The patch-clamp technique was used to identify BKCa and to study their modulation by bFGF in cultured endothelial cells of human umbilical cord veins (HUVEC). Cell counts of HUVEC were carried out on different days to analyze bFGF-induced cell proliferation and its influence by the specific BKCa blocker iberiotoxin (IBX). Using single-channel recordings, we found characteristic BKCa with a single-channel slope conductance of 170.3 ± 2.1 pS (n = 7), half-maximal activation at internal pCa = 5.7 (n = 5; test potential: 80 mV), and dose-dependent block by IBX (25–100 nmol/l). In cell-attached patches bFGF (50 ng/ml) caused a significant increase in the open-state probability (NPo) after 6 min at test potentials of 80 and 100 mV (n = 28; p < 0.001), respectively, which lasted up to 30 min. After preincubation with pertussis toxin (100 ng/ml; 4 h) bFGF superfusion did not cause a significant increase in BKCa activity until 25 min had passed (n = 20; p < 0.01). Addition of 100 nmol/l IBX to the pipette solution caused a total block of BKCa within 2 min in cell-attached patches, whereas bFGF (50 ng/ml) was not able to activate BKCa. When incubated with IBX (25–100 nmol/l) every 2 days, bFGF-induced proliferation of HUVEC was significantly decreased by 50 (–41%) and 100 nmol/l (–50%) IBX (n = 5; p < 0.001) after 7 days. We conclude that activation of BKCa by bFGF may play an important role in bFGF-induced proliferation of human endothelial cells and thus might be important in the process of angiogenesis and vascular remodelling.

Dose-dependent activation of Ca2+-activated K+ channels by ethanol contributes to improved endothelial cell functions

BACKGROUND Regular moderate alcohol (EtOH) intake seems to protect against both coronary artery disease and ischemic stroke, whereas the risk increases with heavy EtOH consumption. Effects of EtOH on endothelial cell function may be relevant to these disparate effects. Potassium channels play an important role in the regulation of endothelial cell functions. Therefore, we investigated whether Ca-activated K channels (BKCa) are modulated by EtOH. Furthermore, we examined whether EtOH-induced changes of endothelial nitric oxide (NO) formation and cell proliferation are due to BKCa activation. METHODS The patch-clamp technique was used to investigate BKCa activity in cultured human umbilical vein endothelial cells (HUVEC). NO formation was analyzed by using the fluorescence dye 4,5-diaminofluorescein. Endothelial proliferation was examined by using cell counts and measuring [H]thymidine incorporation. RESULTS EtOH dose-dependently (10-150 mmol/liter) modulated BKCa-activity, with the highest increase of open-state probability at a concentration of 50 mmol/liter (n = 13; p < 0.05). Inside-out recordings revealed that this effect was due to direct BKCa activation, whereas open-state probability was not changed in cell-attached recordings after pertussis toxin preincubation. EtOH (10 and 50 mmol/liter) caused a significant increase of NO levels, which was blocked by the highly selective BKCa inhibitor iberiotoxin (100 nmol/l; n = 30; p < 0.05). Higher concentrations of EtOH (100 and 150 mmol/liter) significantly reduced NO synthesis (n = 30; p < 0.05). Both methods revealed a significant increase of HUVEC proliferation, which was inhibited by iberiotoxin (n = 30; p < 0.05). At a concentration of 150 mmol/liter, EtOH caused a significant reduction of endothelial proliferation. CONCLUSIONS EtOH directly activates BKCa in HUVEC, leading to an increase of endothelial proliferation and production of NO. These results indicate a possible beneficial effect of low-dose EtOH on endothelial function, whereas higher concentrations must be considered as harmful.

Cerivastatin Activates Endothelial Calcium–Activated Potassium Channels and Thereby Modulates Endothelial Nitric Oxide Production and Cell Proliferation

Statins are known to counteract the process of arteriosclerosis by exerting direct pleiotropic effects on vascular endothelium. The aim of this study was to investigate a possible effect of cerivastatin on endothelial Ca(2+)-activated K+ channels (BK(Ca)) and to assess their contribution to cerivastatin-mediated changes of endothelial nitric oxide (NO) production and proliferation. Membrane potential was measured using bis-1,3-dibutylbarbituric acid-trimethine oxonol-fluorescence imaging. Patch-clamp recordings of BK(Ca) were performed on cultured human umbilical vein endothelial cells. NO production was measured using 4,5-diaminofluorescein-fluorescence imaging and a [(3)H]cGMP RIA. Proliferation was analyzed by means of cell counts and [(3)H]thymidine incorporation (TI). Cerivastatin (0.001 to 0.05 micromol/L) caused a significant membrane hyperpolarization (n = 30; P < 0.05). This effect was abolished using the BK(Ca) inhibitor iberiotoxin (IBX; 100 nmol/L). The addition of mevalonate (500 micromol/L) blocked the BK(Ca) activation induced by cerivastatin (n = 19; P < 0.05). Endothelial cGMP level was increased by acetylcholine (ACh; 1 micromol/L). The combination of ACh and cerivastatin additionally increased cGMP levels, with a maximum at 0.03 micromol/L cerivastatin (84%; n = 10, P < 0.01). ACh-induced increase of cGMP-level was significantly reduced by IBX (n = 10, P < 0.01) as it was with all combined administrations of ACh and cerivastatin. 4,5-Diaminofluorescein-fluorescence measurements revealed a significant increase of NO levels by cerivastatin, which was abolished by IBX (n = 30; P < 0.05). Cell counts and TI demonstrated significant inhibition of human umbilical vein endothelial cell proliferation with a maximum at 0.03 micro mol/L (cell count, -32.2%; TI, -70%; n = 12; P < 0.01). These data show that cerivastatin activates endothelial BK(Ca), which plays an important role in the signaling of cerivastatin-mediated endothelial NO production and proliferation.

Prävalenz und Bedeutung der koronaren Kollateralzirkulation bei Patienten mit akutem Myokardinfarkt

Angiograms from consecutive and unselected patients with acute myocardial infarction were studied with respect to the prevalence as well as the significance of coronary collateral circulation to myocardium distal to the acute coronary occlusion. Methods Coronary angiograms were obtained from 700 consecutive and unselected patients with an acute transmural infarction within 3.7±3 hours (0.5–12) of symptom onset. No patient had undergone iv thrombolysis prior to angiography. Complete and acute vessel occlusion was found in 626/700 patients (89%). Coronary collaterals were detected and graded using Rentrop’s classification. The grade of collateral circulation was related to the clinical course after 30 days and to the global and regional left ventricular wall motion. Results Collaterals were found in 334 patients (69%); 242 patients (38%) had collateral flow grade 2 or 3. Collaterals were demonstrated more frequently in women vs men and in patients with multivessel disease. The prevalence of collaterals was unrelated to age and the presence of diabetes mellitus. Patients who had angiography within 3 hours of symptom onset had collaterals detected less frequently than patients who had angiography beyond 6 hours (66% vs 75%, p<0.05). No collaterals were found in 17/37 patients (47%) in cardiogenic shock and inferior MI but in only 30/164 patients (18%, p<0.01) without shock. Global and regional left ventricular wall motion after 2 weeks was unrelated to the degree of coronary collateral circulation during acute myocardial infarction. Conclusion Collateral circulation to myocardium distal to an acutely occluded coronary artery is detected in 2/3 patients during the acute infarct phase. The absence of collaterals is related to the early occurrence of cardiogenic shock in patients with inferior MI but not to the presence of diabetes mellitus. After direct angioplasty of the infarct vessel, the protective effects of coronary collaterals on chronic LV function remain uncertain. Um Prävalenz und klinische Bedeutung der Kollateralzirkulation zu Myokard distal eines akuten Koronarverschlusses zu untersuchen, wurden Koronarangiogramme von konsekutiven und unselektierten Patienten mit akutem Myokardinfarkt vor jeglicher Reperfusionstherapie analysiert und mit klinischen Daten korreliert. Methodik Bei 700 konsekutiven und unselektierten Patienten mit akutem ST-Hebungsinfarkt wurde im Mittel nach 3,7±3 Stunden (0,5–12) ein Koronarangiogramm ohne vorangehenden Thrombolyseversuch angefertigt. Bei 626/700 Patienten (89%) lag ein kompletter Koronargefäßverschluss vor. Bei diesen Patienten wurde die Kollateralzirkulation klassifiziert. Der Kollateralisierungsgrad wurde mit klinischen Befunden, dem Verlauf nach 30 Tagen und der globalen und regionalen Wandbewegung korreliert. Ergebnisse Kollateralen waren bei 334/626 Patienten (69%) nachweisbar, bei 242 Patienten (38%) ein Kollateralfluss Grad 2 oder 3. Bei Frauen gegenüber Männern und bei Patienten mit einer Mehrgefäßerkrankung wurden häufiger Kollaterale gefunden. Die Prävalenz von Kollateralen war von Alter und Diabetes mellitus unabhängig. Patienten, die innerhalb von 3 Stunden nach Symptombeginn angiographiert werden konnten, hatten weniger häufig Kollateralen als nach >>;6 Stunden angiographierte Patienten (66% vs. 75%, p<0,05). Von 37 Patienten mit kardiogenem Schock bei Hinterwandinfarkt hatten 17 (46%) keine Kollateralzirkulation gegenüber 30/164 Personen ohne Schock (18%, p<0,01). Die nach etwa 2Wochen angiographisch bestimmte globale und regionale LV-Funktion zeigte keine Abhängigkeit von der Kollateralisierung. Zusammenfassung Eine Kollateralzirkulation zu Myokard distal eines akuten Koronarverschlusses ist angiographisch bei etwa 2/3 der Patienten schon früh nach Infarktbeginn nachweisbar. Die Abwesenheit von Kollateralen ist bei Patienten mit Hinterwandinfarkt mit dem frühen Auftreten eines kardiogenen Schocks assoziiert, nicht aber dem Vorhandensein eines Diabetes mellitus. Ein protektiver Effekt von Kollateralen bezüglich der chronischen LV-Funktion lässt sich nach direkter Angioplastie des Infarktgefässes nicht nachweisen.

Basic Fibroblast Growth Factor–Induced Endothelial Proliferation and NO Synthesis Involves Inward Rectifier K+ Current

Objectives—Inward rectifier K+ currents (Kir) determine the resting membrane potential and thereby modulate essential Ca2+-dependent pathways, like cell growth and synthesis of vasoactive agents in endothelial cells. Basic fibroblast growth factor (bFGF) acts as a vasodilatator and angiogenic factor. Therefore, we investigated the effect of bFGF on Kir and assessed the role in proliferation and nitric oxide (NO) formation of endothelial cells. Methods and Results—Using the patch-clamp technique, we found characteristic Kir in human umbilical cord vein endothelial cells (HUVEC), which were dose-dependently blocked by barium (10 to 100 μmol/L). Perfusion with bFGF (50 ng/mL) caused a significant increase of Kir, which was blocked by 100 μmol/L barium (n=18, P <0.01). The bFGF-induced HUVEC proliferation was significantly inhibited when using 50 to 100 μmol/L barium (n=6; P <0.01). NO production was examined using a cGMP radioimmunoassay. bFGF caused a significant increase of cGMP levels (n =10; P <0.05), which were blocked by barium. Conclusions—Modulation of Kir plays an important role in bFGF-mediated endothelial cell growth and NO formation.

Clinical performance of a specific algorithm to reconfirm self-terminating ventricular arrhythmias in current implantable cardioverter-defibrillators

Inappropriate shock therapy is a frequent problem in patients with implantable cardioverter-defibrillators (ICDs), caused mostly by supraventricular rhythms. Self-terminating ventricular arrhythmias (STVAs), however, may also lead to inappropriate shock discharges even in ICDs with abortive shock capabilities. The aim of this study was to evaluate the clinical performance of a specific ventricular tachycardia/ventricular fibrillation (VT/VF) reconfirmation algorithm implemented in current ICD devices from Medtronic to prevent inappropriate shock discharges due to STVAs. A total of 161 STVA episodes were documented in 59 of 150 patients (39%) within a mean follow-up of 30 +/- 20 months and resulted in 25 inappropriate shock discharges in 15 of 150 patients (10%) despite activation of the reconfirmation algorithm. The first synchronization interval of the algorithm was met in 92% of STVA episodes with and even 38% of STVA episodes without shock delivery. A reduced incidence of inappropriate shocks due to STVAs was found with tachycardia/fibrillation detection intervals (TDI/FDI) programmed to shorter cycle lengths < or =280 ms or the use of the first 2 cycles after the end of charging to be considered for reconfirmation only. Thus, inappropriate shocks due to STVAs still occur in 10% of patients with ICDs despite activation of a specific VT/VF reconfirmation algorithm, and are mainly caused by meeting the first synchronization interval that therefore should be shortened in cycle length. Moreover, to reduce the likelihood of inappropriate shocks, the VF reconfirmation algorithm should be optimized by basing the synchronization intervals exclusively on the FDI with short cycle lengths or using the first 2 cycles for reconfirmation only.