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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.
ABSTRACT
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.
The effect of bFGF on inward rectifier K+ currents (Kir) was analyzed in HUVEC. bFGF caused an increase of Kir, which was blocked by barium. Endothelial proliferation and NO production induced by bFGF were inhibited by reducing Kir activity with barium.
Key Words: growth factors ? ion channels ? angiogenesis ? nitric oxide
Introduction
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 (Kir). Changes of the Kir through vasoactive substances like bFGF may therefore be of importance to the endothelial regulatory functions. A modulation of Kir 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 Kir was observed under the influence of shear stress.14 At present, little is known about changes of Kir 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 (BKCa) have been directly associated with the regulation of NO synthesis.23–25
The aim of our study, therefore, was to determine whether Kir is modulated by bFGF and to assess the role of Kir modulation in bFGF-mediated proliferation and NO synthesis of human endothelial cells.
Methods
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 Kir 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
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 Kir, 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 (EK: –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 Kir. 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 Kir. 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 Kir 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
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 Kir (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 Kir is activated by bFGF.
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 Kir modulation has a role in the bFGF-mediated endothelial cell growth, we tested whether a blockade of Kir 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 Kir 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.
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 -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 Kir 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 Kir in bFGF-regulated NO production. The results are summarized in Figure 4.
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
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 Kir 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 Kir 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 Kir and, most importantly, 100 μmol/L barium completely blocked Kir, which is in line with findings of other working groups.8,29 Although barium is not a selective blocker of Kir, this very low concentration of 100 μmol/L barium is relatively selective for Kir, 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 Kir, 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 Kir 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 Kir 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 Kir completely, caused a significant inhibition of bFGF-mediated cell growth. Activation of Kir 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 Kir 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 Kir. In conclusion, the results of our study show that Kir plays an important role in endothelial proliferation and synthesis of NO caused by bFGF.
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