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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.

Margatoxin Inhibits VEGF-Induced Hyperpolarization, Proliferation and Nitric Oxide Production of Human Endothelial Cells

Background: Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells (EC) in vitro and angiogenesis in vivo. Furthermore, a role of VEGF in K+ channel, nitric oxide (NO) and Ca2+ signaling was reported. We examined whether the K+ channel blocker margatoxin (MTX) influences VEGF-induced signaling in human EC. Methods: Fluorescence imaging was used to analyze changes in the membrane potential (DiBAC), intracellular Ca2+ (FURA-2) and NO (DAF) levels in cultured human EC derived from human umbilical vein EC (HUVEC). Proliferation of HUVEC was examined by cell counts (CC) and [3H]-thymidine incorporation (TI).Results: VEGF (5–50 ng/ml) caused a dose-dependent hyperpolarization of EC, with a maximum at 30 ng/ml (n = 30, p < 0.05). This effect was completely blocked by MTX (5 µmol/l). VEGF caused an increase in transmembrane Ca2+ influx (n = 30, p < 0.05) that was sensitive to MTX and the blocker of transmembrane Ca2+ entry 2-aminoethoxydiphenyl borate (APB, 100 µmol/l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca2+ (n = 30, p < 0.05). HUVEC proliferation, examined by CC and TI, was significantly increased by VEGF and inhibited by MTX (CC: –58%, TI –121%); APB (CC –99%, TI –187%); N-monomethyl-L-arginine (300 µmol/l: CC: –86%, TI –164%). Conclusions: VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca2+ entry that is responsible for the effects on endothelial proliferation and NO production.

The K+-channel opener NS1619 increases endothelial NO-synthesis involving p42/p44 MAP-kinase

Ca(2+)-activated K(+) channels with large conductance (BK(Ca)) have been shown to play an important role in the regulation of vascular tone. We examined the role of the p42/p44 MAP-kinase (p42/p44(MAPK)) on nitric oxide (NO) production in human endothelial cells induced by the BK(Ca)-opener NS1619. Using DiBAC-fluorescence imaging a concentration-dependent (2.5-12.5 microM) hyperpolarization induced by NS1619 was observed. A significant increase of intracellular Ca(2+)-concentration by NS1619 was seen using Fura-2-fluorescence-imaging, which was blocked by 2-APB, or reduction of extracellular Ca(2+) (n=30; p<0.05). A cGMP-radioimmunoassay was used to examine NO synthesis. NS1619 significantly increased cGMP levels which was inhibited by LNMMA, iberiotoxin, BAPTA, 2-APB, reduction of extracellular Ca(2+), PD 98059, or U0126 (cGMP (pmol/mg protein): NS1619 3.25 +/- 0.85; NS1619 + L-NMMA 0.86 +/- 0.02; NS1619 + iberiotoxin 0.99 +/- 0.09; NS1619 + BAPTA 0.93 +/- 0.29; NS1619 + 2-APB 0.99 +/- 0.31; NS1619 + Ca(2+)-reduction 1.17 +/- 0.06; NS1619 + PD98059 1.06 +/- 0.49; NS1619 + U0126 1.10 +/- 0.24; n=10; p<0.05). The phosphorylation of eNOS and p42/p44(MAPK) was examined by immunocytochemistry. Phosphorylation of p42/p44(MAPK) was significantly increased after 10 minutes of NS1619 stimulation, whereas eNOS phosphorylation was not changed over a period of 1 to 30 minutes. NS1619-induced hyperpolarization was not affected by treatment with PD 98059 or U0126. Additionally, NS1619 inhibited endothelial proliferation involving a NO-dependent mechanism. Our data demonstrate that NS1619 causes a transmembrane Ca(2+)-influx leading to an increased NO production involving p42/p44(MAPK). This rise of NO formation is responsible for the NS1619 induced reduction of endothelial cell growth.

Feasibility of everolimus-eluting bioresorbable vascular scaffolds in patients with chronic total occlusion

OBJECTIVE This study evaluates the feasibility of percutaneous coronary intervention with bioresorbable vascular scaffolds (BVSs) in chronic total occlusion (CTO) lesions. BACKGROUND Everolimus-eluting BVSs represent a new approach to treating coronary artery disease, but experience with CTO is limited. METHODS Patients with a previously diagnosed CTO who had been treated with BVS were included. Patients with unsuccessful CTO procedures and patients treated with drug-eluting stents were excluded. Difficulty of the CTO procedure was assessed by the J-score. RESULTS A total of 23 patients were included. Mean age was 60.4 ± 9.0 years, 17.4% were female, 91.3% suffered from hypertension and 34.8% from diabetes. Mean J-score was 1.7 ± 1.0. Median procedure time was 70 min (54-85), mean contrast volume was 213.5 mL (±94.2) and median fluoroscopy time was 19.1 min (13.1-30.0). A total of 64 BVSs were implanted with a mean number of 2.8 ± 1.0 BVSs per patient, a mean total BVS length of 64.8 ± 24.2 mm per lesion, and a mean BVS diameter of 3.1 ± 0.2 mm. Neither a scaffold-related dissection nor any other intra-procedural complication occurred. During a follow-up of 108 (79.5-214.5) days one in-scaffold thrombosis was noted 4 days after the CTO procedure due to a lack of dual antiplatelet therapy. No further major adverse cardiac events occurred. CONCLUSION These results suggest that BVS implantation in CTO lesions can be performed with good procedural success and reasonable clinical short-term outcome in highly selected cases.

Apigenin-induced nitric oxide production involves calcium-activated potassium channels and is responsible for antiangiogenic effects.

Summary.  Background: The dietary flavonoid apigenin (Api) has been demonstrated to exert multiple beneficial effects upon the vascular endothelium. The aim of this study was to examine whether Ca2+‐activated K+ channels (KCa) are involved in endothelial nitric oxide (NO) production and antiangiogenic effects.Methods: Endothelial NO generation was monitored using a cyclic guanosine monophosphate radioimmunoassay. KCa activity and changes of the intracellular Ca2+ concentration [Ca2+]i were analyzed using the fluorescent dyes bis‐barbituric acid oxonol, potassium‐binding benzofuran isophthalate, and fluo‐3. The endothelial angiogenic parameters measured were cell proliferation, [3H]‐thymidine incorporation, and cell migration (scratch assay). Akt phosphorylation was examined using immunohistochemistry.Results: Api caused a concentration‐dependent increase in cyclic guanosine monophosphate levels, with a maximum effect at a concentration of 1 μm. Api‐induced hyperpolarization was blocked by the small and large conductance KCa inhibitors apamin and iberiotoxin, respectively. Furthermore, apamin and iberiotoxin blocked the late, long‐lasting plateau phase of the Api‐induced biphasic increase of [Ca2+]i. Inhibition of Ca2+ signaling and the KCa blockade both blocked NO production. Prevention of all three (NO, Ca2+, and KCa signaling) reversed the antiangiogenic effects of Api under both basal and basic fibroblast growth factor‐induced culture conditions. Basic fibroblast growth factor‐induced Akt phosphorylation was also reduced by Api.Conclusions: Based on our experimental results we propose the following signaling cascade for the effects of Api on endothelial cell signaling. Api activates small and large conductance KCa, leading to a hyperpolarization that is followed by a Ca2+ influx. The increase of [Ca2+]i is responsible for an increased NO production that mediates the antiangiogenic effects of Api via Akt dephosphorylation.

A new signaling mechanism of hepatocyte growth factor‐induced endothelial proliferation

Summary.  Background: The hepatocyte growth factor (HGF) has been shown to promote endothelial cell proliferation. In this study, the signaling cascade responsible for the HGF‐induced proliferation was examined. Methods: The proliferation of human umbilical cord vein endothelial cells (HUCVEC) was determined using cell counts. Changes of the membrane potential were analyzed using the fluorescence dye DiBAC. Intracellular cGMP‐levels were measured by means of [3H]‐cGMP‐radioimmunoassay. Phosphorylation of the p42/p44 MAP‐kinase (MAPK) and the endothelial nitric oxide synthase (eNOS) was analyzed by immunocytochemistry. Results: A dose‐dependent (1–30 ng mL−1) increase of HUCVEC proliferation with a maximum at a concentration of 15 ng mL−1 was induced by HGF. This effect was significantly reduced by the addition of the K+ channel blocker iberiotoxin (100 nmol L−1), eNOS inhibitor l‐NMMA (300 μmol L−1), or the MEK inhibitor PD 98059 (20 μmol L−1). A HGF‐induced hyperpolarization that was blocked by iberiotoxin was observed. In addition, HGF‐induced activation of the eNOS was blocked by the K+ channel inhibitor. An increase of +101% MAPK phosphorylation was induced by HGF, which was blocked, if the cells were treated with l‐NMMA (n = 20; P < 0.05), whereas HGF‐induced phosphorylation of the eNOS was not affected by MEK inhibition. Conclusions: Hepatocyte growth factor modulates endothelial K+ channels causing an activation of the eNOS; the increase of nitric oxide is necessary for the phosphorylation of the MAPK inducing the proliferation of HUCVEC.

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.

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 (Ba2+)-sensitive potassium channels (KiCa) 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 µg/ml. This effect of LPS was completely abolished in the presence of Ba2+ (100 µmol/l). In addition, LPS-induced proliferation was significantly reduced by Ba 2+ (control, 100%; LPS 5 µg/ml, 175%; LPS + Ba2+ 100 µmol/l, 136%; n = 12, P < 0.05). To examine whether KiCa are activated by LPS, changes of U937 membrane potential were determined. LPS (5 µg/ml) caused a hyperpolarization of U937 cells indicating a flux of K+ ions out of the cells. This effect was completely blocked by Ba2+ (100 µmol/l). In conclusion, we demonstrate that LPS activates KiCa in U937 cells, which is responsible for LPS-induced adhesion of these cells to endothelial cells, and to the proliferation of U937 cells.

Baroreceptor stimulation in a patient with preexisting subcutaneous implantable cardioverter defibrillator: WEIPERT et al.

Many patients with severe heart failure (HF) have an indication for baroreflex activation therapy (BAT) and an implantable cardioverter‐defibrillator (ICD). Concerns about device‐device interactions were addressed in a study with small sample size that concluded combined BAT and ICD therapy is safe. There are no published data, however, concerning device‐device interactions between BAT and a subcutaneous ICD (S‐ICD). Since BAT frequently interferes with surface electrocardiogram recordings, there are doubts about compatibility of BAT and S‐ICD devices.

Barostim Implantation with Ipsilateral Carotid Endarterectomy as a One-Stage Procedure

Clinical trials have demonstrated significant and lasting reductions in arterial pressure from baroreflex activation therapy (BAT), resulting from electrical stimulation of the carotid sinus in patients with resistant arterial hypertension. Significant carotid atherosclerosis, however, has been a contraindication for ipsilateral implantation due to a potentially increased risk of periprocedural stroke and uncertain antihypertensive efficacy. Here, we describe the first case in which BAT was applied safely and effectively in a patient with distinct cerebral arteriosclerosis after ipsilateral carotid endarterectomy as a one-stage procedure without neurologic complications. BAT resulted in satisfactory blood pressure levels despite distinct cerebral atherosclerosis after an 18-month follow-up period.