This Article Abstract Full Text (PDF) All Versions of this Article: 24/7/1229    most recent 01.ATV.0000130663.37663.6av1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Scharbrodt, W. Articles by Wiecha, J. Search for Related Content PubMed PubMed Citation Articles by Scharbrodt, W. Articles by Wiecha, J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB BARIUM COMPOUNDS BARIUM, ELEMENTAL CALCIUM COMPOUNDS CALCIUM, ELEMENTAL NITRIC OXIDE POTASSIUM Related Collections Angiogenesis Cell signalling/signal transduction Growth factors/cytokines Ion channels/membrane transport Endothelium/vascular type/nitric oxide

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:1229.)
© 2004 American Heart Association, Inc.

Vascular Biology

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

Wolfram Scharbrodt; Christoph Rüdiger Wolfram Kuhlmann; Yongijan Wu; Christian Alexander Schaefer; Astrid Kerstin Most; Ulrich Backenköhler; Thomas Neumann; Harald Tillmanns; Bernd Waldecker; Ali Erdogan; Johannes Wiecha

From the Department of Cardiology and Angiology (W.S., C.R.W.K., Y.W., C.A.S., A.K.M., U.B., T.N., H.T., B.W., A.E.), Justus-Liebig-University of Giessen, Germany; and the Department of Internal Medicine (J.W.), Hospital Bad Orb, Germany.

Correspondence to Christoph Rüdiger Wolfram Kuhlmann, Justus-Liebig University Giessen, Department of Cardiology and Angiology, Klinikstr. 36, 35392 Giessen, Germany. E-mail Christoph.R.Kuhlmann{at}innere.med.uni-giessen.de

	Abstract

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Objectives— 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 µmol/L). Perfusion with bFGF (50 ng/mL) caused a significant increase of K_ir, 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 K_ir plays an important role in bFGF-mediated endothelial cell growth and NO formation.

The effect of bFGF on inward rectifier K⁺ currents (K_ir) was analyzed in HUVEC. bFGF caused an increase of K_ir, which was blocked by barium. Endothelial proliferation and NO production induced by bFGF were inhibited by reducing K_ir activity with barium.

Key Words: growth factors • ion channels • angiogenesis • nitric oxide

	Introduction

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Vascular endothelial cells play an essential role in the process of angiogenesis and vessel repair. Basic fibroblast growth factor (bFGF), which is released from endothelial cells and macrophages during hypoxia or vascular injury, takes part in this process by influencing endothelial proliferation and migration.^1–4 In general, it is well-documented that many of the endothelial functions such as the synthesis and release of nitric oxide (NO), the von Willebrand factor, or the regulation of permeability are initiated by Ca²⁺-dependent mechanisms.^5–7 Because endothelial cells lack voltage-dependent Ca²⁺ channels, the membrane potential, which modulates the driving force for transmembrane Ca²⁺ fluxes, is an important regulator of intracellular Ca²⁺ signaling and the functional state of endothelial cells.^8–10 The major ionic current that determines the resting membrane potential is thought to be carried through inward rectifier K⁺ currents (K_ir). Changes of the K_ir through vasoactive substances like bFGF may therefore be of importance to the endothelial regulatory functions. A modulation of K_ir in endothelial cells was already described. Agonists such as angiotensin II, endothelin-1, and histamine inhibit the inward rectifier K⁺ current.^11–13 An activation of K_ir was observed under the influence of shear stress.¹⁴ At present, little is known about changes of K_ir caused by the angiogenic peptide bFGF. There is growing evidence that ion channels are involved in the process of cell proliferation and NO generation. Proliferation was inhibited in human melanoma cells by blockers of delayed rectifier potassium channels, and tamoxifen has been shown to block proliferation and voltage-dependent K⁺ channels in neuroblastoma cells.^15,16 In addition, voltage-dependent gating of the inward-rectifying K⁺ current was linked to the cell-cycle clock.¹⁷ Besides these direct observations in cancer cells, the mitogenic peptide platelet-derived growth factor has been shown to activate nonspecific cation channel mouse fibroblasts.¹⁸ Blockade of this ion channel caused an inhibition of platelet-derived growth factor-induced cell proliferation.¹⁹ Previously, we have shown that an activation of endothelial Ca²⁺-activated K⁺ channels by bFGF is linked to the bFGF-mediated endothelial cell growth.^20,21 In addition, K⁺ channels have been shown to influence endothelium-dependent vasodilatation, because intracellular calcium has been shown to be essential for agonist-induced NO formation.²² Recently, Ca²⁺-activated K⁺ channels of large conductance (BK_Ca) have been directly associated with the regulation of NO synthesis.^23–25

The aim of our study, therefore, was to determine whether K_ir is modulated by bFGF and to assess the role of Kir modulation in bFGF-mediated proliferation and NO synthesis of human endothelial cells.

	Methods

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Cells
Human umbilical cord veins endothelial cells (HUVEC) were isolated as described recently.²⁵ Cells were isolated by a collagenase digestion procedure. The endothelial cell basal medium (PromoCell, Heidelberg, Germany) was enriched with 10% fetal calf serum (PAA, Linz, Austria). The culture medium was changed every 48 hours. All experiments were performed using endothelial cells from subcultures 2 to 6.

Electrophysiology
The patch-clamp technique was applied in the whole-cell mode using a List P/M patch-clamp amplifier (List Electronic).²⁶ Borosilicate fire-polished pipettes (Hilgenberg) had resistances of 1.5 to 2.5 mol/L when filled with the mentioned pipette solution. Membrane currents were filtered with a 6-pole Bessel filter and sampled at a rate of 5 kHz. In all experiments, holding potential was –20 mV. To elicit inward currents, the following voltage protocol was used: 250 ms long steps, ranging from –45 mV to –120 mV and spaced by 15 mV. Electrical stimulation and data acquisition were performed using pCLAMP 6.0.3 (Axon Instruments). To analyze the current, we measured the amplitude 175 ms after the beginning of the voltage pulse. Although we expected a higher deviation of our data, we used the original values, which have not been normalized to the membrane capacity or to the maximum current, for the statistic analysis.

Solutions and Reagents
For electrophysiological studies, HUVEC were maintained in an extracellular (bath) solution containing (in mmol/L): NaCl 140; d-glucose 5.5; HEPES 10; KCl 5; MgCl₂ 0.5; and CaCl₂ 1.5 (pH was adjusted to 7.3 with NaOH). In some experiments 10, 50, and 100 µmol/L barium (Ba²⁺) (Sigma, Deisenhofen, Germany), and/or 50 ng/mL human bFGF (PeproTech, London, UK) were added to the bath solution. The standard pipette solution contained (in mmol/L): K-aspartate 110; KCl 30; MgCl₂ 1; EGTA 0.5; and Na₂ATP 4 (pH was adjusted to 7.2 with KOH). All experiments were performed at room temperature (20°C to 22°C).

Cell Proliferation
For the examination of cell proliferation, HUVEC of confluent primary cultures were trypsinized (0.05% wt/vol trypsin and 5 mmol/L EDTA containing Ca²⁺ free solution) and seeded at a density of 20 000/well (30 cm²). On the first day (day 0), the cells were incubated in the aforementioned basal medium. The following day’s incubation medium was modified by adding 50 ng/mL bFGF and/or different concentrations of barium (10, 50, and 100 µmol/L). The modified medium was replaced every 2 days and counting was performed on day 7. For counting, cells were detached by trypsinizing them, and samples of the mixed cell suspension were transferred 4 times into a Neubauer chamber. For further analysis, the mean values of the 4 counts were used. The number of HUVEC is expressed per well.

cGMP Radioimmunoassay
Endothelial NO production was examined using a cGMP radioimmunoassay kit (cGMP-RIA) (Amersham, Freiburg, Germany). HUVEC were stimulated for 30 minutes with combinations of bFGF (50 ng/mL) and barium (100 µmol/L). Incubation was stopped by the addition of ice-cold ethanol. The cell lysate was centrifuged, and measurements of cGMP levels of the supernatant were performed using the cGMP-RIA.

Statistical Analysis
Statistical significance for repeated measurements of Kir was determined by using a Friedman test (P<0.05; SPSS for Windows; version 5.0.2), and for the following multiple comparisons by means of the Nemenyi test. The dose–response curve to describe the effect of nicotine on K_ir was achieved by fitting the data using a single sigmoidal function. Data of cell proliferation and cGMP measurements were analyzed by ANOVA followed by post hoc Tukey test (SPSS for Windows; version 5.0.2). Results are expressed as mean values±SEM.

	Results

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Inward Rectifier K⁺ Current in HUVEC
To measure inward rectifier K⁺ currents, we applied hyperpolarizing voltage steps from a holding potential of –20 mV to test potentials ranging from –45 mV to –120 mV (steps: –15 mV) to single endothelial cells in the whole-cell configuration of the patch-clamp technique. While using this kind of voltage–clamp protocol, the elicited inward currents enabled us to distinguish mainly between 2 cell types. First, we found HUVEC with a predominant K_ir, which has been described in more detail by others.^9,12,27–29 At –120 mV, the current showed a fast inactivation. The reversal potential of the currents in these cells was –78±8 mV (n=10), which is close to the expected K⁺ equilibrium potential (E_K: –83 mV). These kind of endothelial cells have been called K⁺-type endothelial cells.¹⁰ The second cell group showed inward currents, which differed in their current–voltage relationship compared with the K⁺-type endothelial cells. The reversal potentials of the currents in this cell group were in the range between –40 mV to –30 mV (not shown), suggesting that these endothelial cells have more inward currents than K_ir. Similar results were observed in bovine aortic and pulmonary artery endothelial cells.^8,30 For our investigations, we only used K⁺-type endothelial cells with a predominant K_ir. A typical feature of inward rectifier K⁺ currents in many tissues is a high-affinity block by extracellular barium. To find further proof for the existence of K_ir in HUVEC, we added 10, 50, and 100 µmol/L barium to the bath solution and elicited inward currents in the whole-cell patch-clamp mode. The current–voltage relationships of these experiments are demonstrated in Figure 1. Our recordings revealed a dose-dependent and potential-dependent block of the inward currents. As described by other working groups, inward rectifier K⁺ currents in endothelial cells were completely and reversibly blocked by 100 µmol/L barium.^10,29

View larger version (10K): [in this window] [in a new window]   Figure 1. Blockade of inward rectifier K+ currents by external barium. Steady-state current–voltage relationship derived from whole-cell currents in K+-type EC with 10 µmol/L, 50 µmol/L, and 100 µmol/L barium in the bath solution. Data represent mean±SEM (n=8).

Effects of bFGF on the Inward Rectifier K⁺ Current
Because bFGF is a vasoactive substance that plays an important role in the process of angiogenesis and vascular remodelling, we analyzed the effect of this heparin-binding growth factor on the inward rectifier K⁺ current.^1,4 Application of 50 ng/mL bFGF caused a significant increase of K_ir (n=18; P<0.05) after 3 minutes at test potentials between –90 mV up to –120 mV. The current–voltage relationship of the inward currents before and after bFGF treatment is summarized in Figure 2. To exclude the activation of another inward current by bFGF, we perfused the endothelial cells with a combination of 50 ng/mL bFGF and 100 µmol/L barium. In all of these experiments, 100 µmol/L barium still completely abolished the inward current (n=8; P=NS; not shown). Therefore, it is very unlikely that any other current besides K_ir is activated by bFGF.

View larger version (7K): [in this window] [in a new window]   Figure 2. Effect of bFGF on inward rectifier K+ currents. Current–voltage relationship of Kir before and after 3 minutes of external perfusion with 50 ng/mL bFGF in K+-type EC (n=18; *P<0.05 versus control; mean±SEM).

Blockade of bFGF-Mediated Endothelial Cell Proliferation by Barium
Our electrophysiological studies revealed a significant increase of Kir in K⁺-type endothelial cells by bFGF. To assess whether this bFGF-induced K_ir modulation has a role in the bFGF-mediated endothelial cell growth, we tested whether a blockade of K_ir by barium will influence bFGF-mediated endothelial cell growth. HUVEC initially seeded at a density of 20 000 cells/well were counted on day 7, while exposed to different culture media. In analogy to our electrophysiological studies, we added barium (10 µmol/L, 50 µmol/L, and 100 µmol/L) every 2 days to the standard culture medium. Basic FGF alone caused an expected exponential cell proliferation. The treatment with different concentrations of barium resulted in a dose-dependent reduction of the bFGF-mediated endothelial cell proliferation. At a concentration of 100 µmol/L barium, which has been shown to completely block the bFGF-induced K_ir activation, bFGF-mediated HUVEC proliferation was significantly reduced by 55% (n=6; P<0.01). Furthermore, a concentration of 50 µmol/L barium was sufficient to significantly block bFGF-mediated cell proliferation by 47% (n=6; P<0.01; Figure 3). To exclude a direct cytotoxic effect of barium on HUVEC, 100 µmol/L barium was added to the culture medium without bFGF. Compared with the control group (basal medium, without barium), no changes in cell growth or severe cell death were observed.

View larger version (11K): [in this window] [in a new window]   Figure 3. Inhibition of bFGF-mediated HUVEC proliferation by barium. Endothelial cell growth measured in the absence and in the presence of bFGF (50 ng/mL), and with different concentrations of barium (Ba). Number of cells are expressed per well (n=6; *P<0.01 versus control; #P<0.01 versus bFGF).

Inhibition of bFGF-Induced cGMP Levels by Barium
The effect of bFGF on endothelial NO synthesis was measured by means of [³H]-cGMP-RIA. Endothelial cGMP levels were significantly increased from 66.7±38.07 (control) to 704.9±33.49 (bFGF) when 50 ng/mL bFGF was added. When K_ir was blocked using barium (100 µmol/L), the bFGF-induced increase of cGMP level was significantly reduced (n=10; P<0.05), demonstrating a significant involvement of K_ir in bFGF-regulated NO production. The results are summarized in Figure 4.

View larger version (7K): [in this window] [in a new window]   Figure 4. Barium blocks bFGF-induced cGMP levels. Endothelial cGMP levels are significantly increased by bFGF (50 ng/mL) compared with the control group (n=10; *P<0.05 versus control). Addition of barium (Ba) (100 µmol/L) reduced bFGF-induced cGMP levels significantly (n=10; #P<0.05 versus bFGF). cGMP levels shown in pmol/well as means±SEM.

	Discussion

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The aim of this study was to prove whether endothelial inward rectifier K⁺ current is modulated by the angiogenic growth factor bFGF. If this is the case, it will be of further interest whether a modulation of this ion current may be involved in bFGF-mediated proliferation and NO production of endothelial cells. Using HUVEC, our voltage-clamp protocol revealed a bimodal distribution of inward currents, similar to a bimodal distribution of resting membrane potential in endothelial cells, which was described by Voets et al and Mehrke et al.^8,30 First, we found a cell group with a predominant inward rectifier K⁺ current, which has already been identified and characterized in more detail by other working groups.^9,11,13,23 The reversal potential of these currents was –78 mV, which is close to the expected K⁺ equilibrium potential. Therefore, these HUVEC have been classified as K⁺-type endothelial cells.¹⁰ Secondly, HUVEC revealed a current–voltage relationship of the inward currents with a reversal potential in the range between –40 mV and –30 mV, suggesting that other currents besides K_ir contribute to the inward currents. Voets et al and Himmel et al have shown that mainly a chloride current and a nonspecific cation current are responsible for this characteristic current–voltage relationship.^8,9 Because we were interested in the inward rectifier K⁺ current, however, our further electrophysiological studies were performed using only K⁺-type EC. For further validation of K_ir in our K⁺-type EC, we applied barium, which is known to cause a high-affinity block of inward rectifier K⁺ current. We found a dose-dependent and voltage-dependent block of K_ir and, most importantly, 100 µmol/L barium completely blocked K_ir, which is in line with findings of other working groups.^8,29 Although barium is not a selective blocker of K_ir, this very low concentration of 100 µmol/L barium is relatively selective for K_ir, because an effective block of other potassium currents such as ATP-sensitive K⁺ currents and Ca²⁺-activated K⁺ currents require a higher dose of barium.^31,32

When applying bFGF to K⁺-type endothelial cells, a significant increase of the inward current was observed. To ensure that bFGF really activates K_ir, we simultaneously perfused HUVEC with bFGF and 100 µmol/L barium and still observed a complete block of the inward currents. Further investigations to analyze the exact signal transduction pathway of K_ir activation after the binding of bFGF to their specific receptors have not been performed. The finding of the activation of Kir by bFGF in K⁺-type endothelial cells raises the question whether this electrophysiological event is one of the early steps in the bFGF-mediated effects on endothelial cells. To test this hypothesis, we performed proliferation studies using the same concentrations of bFGF and barium that were used in the electrophysiological studies. The resulting data suggest a close correlation between bFGF-mediated endothelial cell growth and K_ir activation. In analogy to the electrophysiological studies, barium caused a dose-dependent block of bFGF-mediated cell proliferation. Applying 100 µmol/L barium, which had blocked K_ir completely, caused a significant inhibition of bFGF-mediated cell growth. Activation of K_ir is certainly not the only link between bFGF receptor activation and the mitogenic response.^4,33 A direct cytotoxic effect of the concentration of barium we used seems to be very unlikely, because a comparison of the cell number in wells containing 100 µmol/L barium to those containing the basal medium (control) revealed no difference. Taken together, our data substantiate the hypothesis that an activation of endothelial inward rectifier K⁺ current seems to be one important early step in the bFGF-mediated endothelial cell proliferation. Further evidence supports the hypothesis that ionic currents are involved in the regulation of cell proliferation, as well as in growth factor-induced cell proliferation. Blockers of volume-sensitive Cl^– channels have been shown to suppress the growth of endothelial cells. The proliferation of human melanoma cells was inhibited in the presence of blockers of delayed rectifier potassium channels. The blockade of bFGF-modulated Ca²⁺-activated K⁺ channels caused an inhibition of bFGF-mediated endothelial cell growth.^15,21,34 Our finding that the bFGF-induced activation of inward rectifier K⁺ currents contributes to the bFGF-mediated proliferation of endothelial cells may provide a signaling pathway that influences angiogenesis. Previous studies have demonstrated that bFGF works as a vasodilatating factor. Meurice et al have shown an improvement of endothelium-dependent vasodilatation induced by bFGF using a hypercholesterolemic and balloon injury rabbit model.^35,36 Identical results were reported by Tiefenbacher et al using arterioles from pig hearts.³⁷ On the cellular level, it was observed that these findings might be explained by an increase of endothelial NO production caused by bFGF.³⁸ Recently, our working group was able to show that K⁺ channels play an important role in acetylcholine-induced NO synthesis.²⁵ To investigate whether bFGF-induced NO generation is influenced by K_ir activity, cGMP levels were measured in the presence and absence of barium (100 µmol/L). Our results clearly demonstrate that bFGF-induced increases of cGMP levels involve K_ir. In conclusion, the results of our study show that K_ir plays an important role in endothelial proliferation and synthesis of NO caused by bFGF.

Received March 25, 2004; accepted April 21, 2004.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 24/7/1229    most recent 01.ATV.0000130663.37663.6av1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Scharbrodt, W. Articles by Wiecha, J. Search for Related Content PubMed PubMed Citation Articles by Scharbrodt, W. Articles by Wiecha, J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB BARIUM COMPOUNDS BARIUM, ELEMENTAL CALCIUM COMPOUNDS CALCIUM, ELEMENTAL NITRIC OXIDE POTASSIUM Related Collections Angiogenesis Cell signalling/signal transduction Growth factors/cytokines Ion channels/membrane transport Endothelium/vascular type/nitric oxide