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.

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 Ca(2+) 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 Ca(2+) (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 [(3)H]-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 micromol/l). VEGF caused an increase in transmembrane Ca(2+) influx (n=30, p<0.05) that was sensitive to MTX and the blocker of transmembrane Ca(2+) entry 2-aminoethoxydiphenyl borate (APB, 100 micromol/l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca(2+) (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 micromol/l: CC: -86%, TI --164%). CONCLUSIONS: VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca(2+) entry that is responsible for the effects on endothelial proliferation and NO production.

Discordant effects of nicotine on endothelial cell proliferation, migration, and the inward rectifier potassium current.

The inward rectifier K+ current (K(ir)) determines the resting membrane potential of endothelial cells. Basic fibroblast growth factor (bFGF) has been shown to activate K(ir) and acts as angiogenic factor and vasodilator. In contrast, nicotine has been demonstrated to reduce endothelium-dependent vasorelaxation by increasing radical formation. Aim of the present study was to investigate whether nicotine modulates K(ir) and if this plays a role in bFGF-mediated proliferation, migration and nitric oxide (NO)-formation of endothelial cells. Using the patch-clamp technique in cultured endothelial cells of human umbilical cord veins (HUVEC), we found characteristic K(ir), which were blocked by extracellular barium (100 micromol/l). Perfusion with nicotine (1 nmol/l-10 micromol/l) revealed a dose-dependent reduction of K(ir). The simultaneous perfusion with bFGF (50 ng/ml) and nicotine (10 micromol/l) still significantly reduced K(ir) (n = 8; P < 0.01). Cell counts revealed that bFGF-mediated proliferation of HUVEC was significantly inhibited when using 1-10 micromol/l nicotine (n = 8, P < 0.01). The bFGF-induced endothelial cell migration--examined using the "Fences-Migration-Assay"--was significantly reduced by 10 mumol/l nicotine (n = 12; P < 0.05). NO-production was examined using a cGMP-Radioimmunoassay. The significant bFGF-induced increase of cGMP-levels was reduced by nicotine (n = 10; P < 0.05). Our data indicate that the modulation of K(ir) seems to be an essential pathway in the antagonistic effects of nicotine on bFGF-mediated endothelial cell growth, migration and NO-formation.

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.

Statins prevent oxidized low-density lipoprotein- and lysophosphatidylcholine-induced proliferation of human endothelial cells.

The proliferation of endothelial cells is induced by oxidized low-density lipoprotein (oxLDL) and its major component, lysophosphatidylcholine (LPC). The aim of this study was to investigate the effect of statins on the proliferation of endothelial cells derived from human umbilical cord veins (HUVEC). Cerivastatin, simvastatin and fluvastatin caused a dose-dependent inhibition of endothelial cell growth (n=12; P<.01) when using cell counts and [3H]-thymidine incorporation, respectively. The strongest inhibition of HUVEC proliferation was achieved at statin concentrations of 0.1 micromol/l (cerivastatin), 2.5 micromol/l (simvastatin) and 1 micromol/l (fluvastatin). Cell counts were significantly reduced from 22937+/-280.6 (control) to 7791+/-133.6 (cerivastatin), 7292+/-146.6 (simvastatin) and 6792+/-135.5 (fluvastatin) (n=12; P<.01). Interestingly, cell proliferation induced by oxLDL (10 microg/ml) and LPC (20 micromol/l) could be effectively prevented using statins at concentrations between 0.01 and 0.1 micromol/l (cerivastatin), 1 and 2.5 micromol/l (simvastatin) and 0.25 and 1 micromol/l (fluvastatin). This effect of the statins was abolished by preincubation with mevalonate (500 micromol/l). Our results demonstrate an interesting direct effect of statins on the proliferation of human endothelial cells induced by oxLDL and LPC, which may be beneficial to prevent vascular effects of these atherogenic lipids.

Basic fibroblast growth factor-induced endothelial proliferation and NO synthesis involves inward rectifier K+ current.

OBJECTIVE: Inward rectifier K+ currents (K(ir)) 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 K(ir) 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 K(ir) in human umbilical cord vein endothelial cells (HUVEC), which were dose-dependently blocked by barium (10 to 100 micromol/L). Perfusion with bFGF (50 ng/mL) caused a significant increase of K(ir), which was blocked by 100 micromol/L barium (n=18, P<0.01). The bFGF-induced HUVEC proliferation was significantly inhibited when using 50 to 100 micromol/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 K(ir) plays an important role in bFGF-mediated endothelial cell growth and NO formation.