Activation of Ca2+ -activated potassium channels is involved in lysophosphatidylcholine-induced monocyte adhesion to endothelial cells.

OBJECTIVE: Ca(2+)-activated K(+)-channels (BK(Ca)) play an important role in lysophosphatidylcholine (LPC)-induced endothelial dysfunction. Aim of our study was to investigate whether LPC-induced activation of BK(Ca) is also involved in monocyte adhesion to endothelial cells (EC). METHODS AND RESULTS: Measurement of membrane potential (MP) was performed using the fluorescence dye DiBAC. Adhesion of the monocytotic cell line U937 to EC was analysed by (3)[H]-thymidine-adhesion-assay. Expression of ICAM-1 and VCAM-1 were analyzed by FACS. LPC induced a hyperpolarization of EC in a dose-dependent manner with the maximum seen with 2 microM. This was prevented by the BK(Ca)-inhibitor iberiotoxin (IBX, 100nM). Adhesion of U937 cells to EC was increased after stimulation of EC with LPC. This effect was time-dependent with the maximum seen after 4h. LPC-induced adhesion was significantly reduced when EC were co-incubated with IBX, or NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI, 5 microM) and also blocked by addition of 2-aminoethoxydiphenylborate (2-APB, 100 microM) or the calcium-chelator BAPTA (10 microM). Stimulation of U937 cells with LPC did not result in an increased adhesion to unstimulated EC. CONCLUSION: Activation of the endothelial BK(Ca) plays an important role in monocyte adhesion to endothelial cells.

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.

Statins inhibit hypoxia-induced endothelial proliferation by preventing calcium-induced ROS formation.

Pathological hypoxia plays an important role in many diseases, such as atherosclerosis, cancer, and rheumatoid arthritis. The aim of the present study was to examine the effects of different statins on hypoxia-induced endothelial cell signalling. Human umbilical cord vein endothelial cells (HUVEC) were treated with NaCN (CN, 2.5 mmol/l) to simulate a transient hypoxia. The CN-induced increase of endothelial cell numbers was significantly (n = 10, p < 0.01) reduced by the Ca(2+) chelator BAPTA (10 micromol/l), or the reactive oxygen species (ROS) scavenger N-acetylcysteine (ACC, 1 mmol/l), or the NAD(P)H-oxidase inhibitor diphenyleneiodonium (DPI, 5 micromol/l). In detail, cell numbers were (in percentage of control): 163.24 (CN), 90.06 (CN+ACC), 92.06 (CN+DPI). Intracellular-Ca(2+) and -ROS, analysed by fluorescence imaging, were significantly increased by CN. Interestingly, the CN-induced increase of ROS was in part Ca(2+)-dependent, whereas the Ca(2+) increase was not ROS-dependent. Simvastatin (5 micromol/l), fluvastatin (2.5 micromol/l), and cerivastatin (0.1 micromol/l) all reduced CN-induced proliferation, ROS generation and Ca(2+) increase. Cell viability was not reduced by the statins and the antiproliferative effect was completely reversed by mevalonate (500 micromol/l). In conclusion our study demonstrates that statins block hypoxia-associated endothelial proliferation by preventing the increase of Ca(2+) and ROS.

Sildenafil inhibits the proliferation of cultured human endothelial cells.

The proliferation of endothelial cells plays a crucial role in the development of intraplaque angiogenesis (IPA). IPA is a major source of intraplaque hemorrhage and therefore contributes to the destabilization of atherosclerotic plaques. Therefore, the aim of the present study was to examine, whether sildenafil inhibits endothelial cell growth. The proliferation of human endothelial cells derived from umbilical cord veins (HUVEC) was examined on DNA level by measurements of ((3)H)-thymidine incorporation. Cell viability was analyzed using trypan blue staining. The proliferation of cultured human endothelial cells was significantly decreased by 1 µmol/l (-48.4%) and 10 µmol/l (-89.6%) sildenafil (n=10, p<0.05). This was not a cytotoxic effect, because cell viability was only reduced at sildenafil concentrations of 50 µmol/l or greater. In addition sildenafil significantly reduced endothelial proliferation induced by bFGF (n=10, p<0.05). The presented results demonstrate an antiangiogenic effect of sildenafil that might be useful in the prevention of atherosclerotic plaque vascularization.

Role of cGMP in sildenafil-induced activation of endothelial Ca2+-activated K+ channels.

Intracellular cGMP is an important second messenger in endothelial cells. Because Ca(2+)-activated K(+) channels with large conductance (BK(Ca)) have been shown to regulate endothelial cell functions, the aim of the present study was to examine whether sildenafil modulates BK(Ca) activity in cultured human endothelial cells. Changes of the endothelial cell membrane potential were analyzed using the fluorescence dye DiBAC. The patch-clamp technique was used to study BK(Ca) in human endothelial cells of umbilical cord veins (HUVEC). Intracellular Ca(2+) levels were analyzed using Fura-2 fluorescence imaging. Sildenafil caused a dose-dependent (0.05-5 micromol/l) hyperpolarization of the endothelial cells with a maximum at a concentration of 1 micromol/l. A significant increase of BK(Ca) activity was induced by sildenafil (1 micromol/l) perfusion. BK(Ca) open state-probability (NPo) was also increased by the cGMP-analogue 8-bromo-cGMP (0.5 mmol/l), whereas inhibition of the cGMP-dependent kinase (PKG) had no effect on NPo. PKG-inhibition abolished 8-bromo-cGMP induced BK(Ca) activation, and reduced sildenafil induced NPo. Furthermore, sildenafil caused a significant increase of intracellular calcium that was blocked by the BK(Ca) inhibitor iberiotoxin (100 nmol/l). In conclusion sildenafil activates BK(Ca) by a mechanism, which involves cGMP. The activation of the BK(Ca) is responsible for the sildenafil-induced increase of intracellular Ca(2+).

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.