Selective Blockade of the Intermediate-Conductance Ca2+-Activated K+ Channel Suppresses Proliferation of Microvascular and Macrovascular Endothelial Cells and Angiogenesis In Vivo
Objective— Ca2+-activated K+ (KCa) channels have been proposed to promote mitogenesis in several cell types. Here, we tested whether the intermediate-conductance KCa channel (IKCa1) and the large-conductance KCa channel (BKCa) contribute to endothelial cell (EC) proliferation and angiogenesis.
Material and Results— Function and expression of IKCa1 and BKCa/Slo were investigated by patch-clamp analysis and real-time RT-PCR in human umbilical vein ECs (HUVECs) and in dermal human microvascular ECs 1 (HMEC-1). HMEC-1 expressed IKCa1 and BKCa/Slo, whereas HUVECs expressed IKCa1. A 48-hour exposure to basic fibroblast growth factor (bFGF) augmented IKCa1 current amplitudes and induced a 3-fold increase in IKCa1 mRNA expression in HUVECs and HMEC-1. Vascular endothelial growth factor (VEGF) was also effective in upregulating IKCa1. BKCa/Slo expression and current amplitudes in HMEC-1 were not altered by bFGF. bFGF- and VEGF-induced EC proliferation was suppressed by charybdotoxin, clotrimazole, or the selective IKCa1 blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), whereas inhibition of BKCa/Slo by iberiotoxin was ineffective. In the Matrigel plug assay in mice, administration of TRAM-34 for 2 weeks significantly suppressed angiogenesis by ≈85%.
Conclusions— bFGF and VEGF upregulate expression of IKCa1 in human ECs. This upregulation of IKCa1 seems to be required for mitogen-induced EC proliferation and angiogenesis in vivo. Selective IKCa1 blocker might be of therapeutic value to prevent tumor angiogenesis.
Angiogenesis is an important process in a variety of physiological and pathophysiological conditions such as embryonic vasculogenesis, wound healing, poststenotic collateral formation, inflammation, and tumor vascularization.1 Regarding the latter, tumor-derived angiogenic factors such as the vascular endothelial growth factor (VEGF) and the basic fibroblast growth factor (bFGF) induce endothelial cell (EC) sprouting, proliferation, migration, and finally new vessel formation.1,2
With respect to EC proliferation, several studies have shown that EC proliferation after stimulation with angiogenic factors is initiated by a rise in intracellular calcium mediated through calcium-influx channels.3–5 By keeping the membrane potential negative, chloride and potassium channels provide the driving force for this calcium entry and thus play an important role in regulating cell cycle progression6–9 as well as angiogenesis, as shown previously for chloride channels.6 In particular, the intermediate-conductance Ca2+-activated K+ (KCa) channel encoded by the IKCa1 gene10,11 is considered an important regulator of cell proliferation because upregulation of this channel has been shown to be an essential step in the mitogenesis of human lymphocytes, rat fibroblast, vascular smooth muscle cells, and cancer cell lines.9,12–17 Expression of intermediate-conductance KCa channel (IKCa1) has been identified in human mesenteric endothelium and arterial ECs of several other species.5,18–20 Besides other KCa channel subtypes such as large-conductance KCa channels (BKCa) and small-conductance KCa channel (SKCa3) identified in human, porcine, and rat ECs,18–21 we showed that IKCa1 in particular plays a predominant role in mediating hyperpolarization in human mesenteric endothelium in situ.18 This endothelial hyperpolarization has been reported to contribute to the generation of endothelial vasodilating factors such as the endothelium-derived hyperpolarizing factor.21,22 Interestingly, in a recent electrophysiological and molecular biological study in situ,18 we observed an increase in IKCa1 expressing ECs in mesenteric arteries from patients with colonic cancer, suggesting that augmentation of endothelial IKCa1 expression may be related to an activation of the endothelium by tumor-derived angiogenic factors and thus EC proliferation and tumor angiogenesis.
In the light of this previous finding, we therefore tested the hypothesis that angiogenic factors modulate IKCa1 expression and function in human ECs and that selective blockade of IKCa1 channels inhibits EC proliferation. We demonstrate that the angiogenic factors VEGF and bFGF induced upregulation of functional IKCa1 expression in human umbilical vein ECs (HUVECs) and human microvascular ECs 1 (HMEC-1). Inhibition of the channel by the IKCa1 blocker clotrimazole (CLT)11 and by the highly selective CLT derivative 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)12 that does not block cytochrome P450 enzymes12 abolished bFGF- or VEGF-induced cell proliferation in vitro and vascularization of Matrigel plugs in vivo. This suggests that IKCa1 controls EC proliferation and angiogenesis, and selective blockade of endothelial IKCa1 may represent a new therapeutic strategy to prevent tumor angiogenesis.
Materials and Methods
Human ECs and In Vitro Proliferation Studies
HUVECs were isolated as described previously.23 HUVECs and HMEC-1, a cell line derived from dermal HMECs24 were cultured as described previously.23 For proliferation and expression studies, HUVECs of second passage were used to avoid senescence.
To induce growth arrest, HUVECs were kept in low serum medium (2% FCS) to allow cell survival for 48 hours before stimulation with bFGF (50 ng/mL) or VEGF-165 (50 ng/mL). HMEC-1 were kept in serum-free medium for 48 hours before stimulation with bFGF (50 ng/mL). At 5% to 10% confluence, photomicrographs of cells were taken in a fixed field before and 48 hours after stimulation and the percentage increase in cell count was calculated for each experiment. For blocker studies, cells were treated with TRAM-34 (0.1 nmol/L to 1 μmol/L),12 the inactive analogue TRAM-7 (1-tritylpyrrolidine; 1 μmol/L),12 CLT (0.1 nmol/L to 1 μmol/L), or iberiotoxin (IbTx; 100 nmol/L).
PD98059, SB203580, and SP600125 were obtained from Tocris. CLT, IbTx, charydotoxin (ChTx), and apamin were obtained from Sigma. bFGF and VEGF-165 were obtained from Biochrom. All other chemicals were obtained from Sigma.
All experiments were conducted in the whole-cell configuration of the patch-clamp technique, and data analysis was performed as described.15,18
RNA Isolation and Quantitative Real-Time RT-PCR
Cell harvest, RNA isolation, quantitative real-time RT-PCR, and evaluation were done as described previously.15 Primer pairs and internal oligonucleotides were: IKCa1 (GenBank accession No. AF022797): F5′-CATCACATTCCTGACCATCG-3′; R5′-ACGTGCTTCTCTGCCTTGTT-3′; and IO 5′-TGGTGACGTGGTGCCGGGC-3′. BKCa (BKCa/Slo; GenBank accession No. NM_002247) were: F5′-GGACTTAGGGGATGGTGGTT-3′; R 5′-AGTGGGAGGAATGGGACAG-3′; and IO 5′-TGCCGACGGACCTGATCTTCTGC-3′. GAPDH (GenBank accession No. BC 013310) were: F 5′-CACCGTCAAGGCTGAGAACG-3′; R 5′-GCCCCACTTGATTTTGGAGG-3′; and IO 5′-CCCATCACCATCTTCCAGGAGCGA-3′.
In Vivo Matrigel Plug Assay
Standard Matrigel (0.5 mL) supplemented with 100 ng/mL bFGF was introduced subcutaneously into the flank of C57/BL6 mice (Charles River Breeding Laboratories). Thereafter, mice were treated daily with TRAM-34 (120 mg/kg IP; n=7) or vehicle (peanut oil; n=6). After 2 weeks, mice were euthanized, and plugs and some surrounding tissue were excised, fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin/eosin (H&E). Animal protocols were approved by the local animal care and use committee. Vascularization of a single plug was determined by counting functional capillaries in randomly chosen cross-sections, and counts of 3 sections were averaged.
Data are given as mean±SE. An unpaired t test was used to calculate differences between groups. P values <0.05 were considered significant.
Upregulation of IKCa1 in Proliferating ECs After Mitogenic Stimulation
To measure alterations of functional KCa channel expression after stimulation with angiogenic factors, we compared whole-cell KCa currents in unstimulated HUVECs and HMEC-1 with those in cells stimulated with bFGF or VEGF for 48 hours.
In HUVECs stimulated with bFGF (50 ng/mL), the KCa current amplitude was much larger than in vehicle-treated cells (Figure 1A). As shown in Figure 1B and 1C, the mean KCa current normalized to cell capacitance was significantly increased after bFGF stimulation. An upregulation of KCa expression was also observed in VEGF (50 ng/mL)-stimulated HUVECs (Figure 1A and 1B; P<0.01). Cell capacitance values were similar in untreated or mitogen-treated cells (untreated 30±4 pF; bFGF 29±5 pF; VEGF 25±3 pF).
The small residual outward KCa current in vehicle-treated HUVECs as well as the upregulated KCa current in mitogen-stimulated HUVECs exhibited voltage independence and slight inward rectification. This current was blocked by the selective IKCa1 blockers TRAM-34, CLT, and ChTx (Figure 1D) with potencies (Kd 7±1 nmol/L for TRAM-34; Kd 25±3 nmol/L for CLT; and Kd 5±1 nmol/L for ChTx; Figure 1E) similar to cloned IKCa1 channels,10,11 native IKCa1 in rat arterial ECs,17 and T lymphocytes.11–13 The inactive analogue TRAM-7,11 the selective BKCa inhibitor IbTx (100 nmol/L), or the selective SKCa blocker apamin (1 μmol/L) had no detectable effect on this current (data not shown). These electrophyiological properties and pharmacological profile indicate that similar to human mesenteric ECs in situ,18 the KCa currents in mitogen-stimulated and -unstimulated HUVECs are mediated exclusively by IKCa1 channels but not by other KCa channels such as BKCa and SKCa channels.
In contrast to HUVECs, HMEC-1 functionally express 2 different KCa currents: a small voltage-independent component seen at negative potentials resembling the IKCa1 current in HUVECs and a large voltage-dependent BKCa current component, steeply increasing at positive membrane potentials (Figure 2A). Forty-eight hours after bFGF stimulation, the composite BKCa/IKCa1 current was increased (Figure 2A). As shown in Figure 2B, the mean KCa current in bFGF-stimulated HMEC-1 (n=21; cell capacitance, 20±1 pF) was significantly increased at holding potentials of −40, 0, and 40 mV, respectively, but not at 100 mV when compared with unstimulated cells (n=10; cell capacitance 23±3 pF). This indicates that similar to HUVECs, the amplitude of the voltage-independent IKCa1 current was increased after bFGF stimulation, whereas the amplitude of the voltage-sensitive BKCa current was not altered by mitogenic stimulation. bFGF or VEGF did not exert any direct modulatory effects on either IKCa1 or BKCa currents under our experimental conditions using a [Ca2+]i of 3 μmol/L for channel activation15,18 (data not shown). This suggests that the increase in IKCa1 current amplitudes observed after mitogenic stimulation most likely reflects a higher quantity of functionally expressed channels in the cell membrane.
The BKCa current was blocked by the BKCa blocker IbTx (Kd 5±1 nmol/L; Figure 2C and 2D), leaving the IKCa1 current component, which was blocked by the IKCa1 blockers TRAM-34 (Kd 6±1 nmol/L; Figure 2E), CLT (Kd 19±2 nmol/L), and ChTx (Kd 5±1 nmol/L), similar to the IKCa1 in HUVECs. The SKCa blocker apamin (1 μmol/L) had no detectable effect (data not shown), indicating that SKCa currents do not contribute to the KCa currents in HMEC-1. This pharmacological profile confirms that in HMEC-1, the KCa currents are composed of a mixture of IKCa1 and BKCa currents.
Alterations in IKCa1 mRNA Expression in Mitogen-Stimulated HUVECs and HMEC-1 Correlate With Changes in Functional Expression
We used a quantitative real-time RT-PCR to determine whether the changes in functional IKCa1 expression after stimulation with bFGF and VEGF were mediated by increases in IKCa1–mRNA levels and thus the result of new protein synthesis. Untreated and stimulated HUVECs contained substantial quantities of IKCa1 mRNA (Figure Ia, available online at http://atvb.ahajournals.org). In keeping with the changes observed in the IKCa1 current amplitudes, the RT-PCR studies revealed a significant 3-fold and 2-fold increase in IKCa1–mRNA levels after 48 hours of bFGF or VEGF stimulation (supplemental Figure Ib). These results indicate that changes in IKCa1–mRNA levels after stimulation underlie the observed changes in IKCa1 currents in HUVECs. Untreated or stimulated HMEC-1 that functionally express BKCa and IKCa1 (Figure 2A through 2C) contained substantial quantities of Slo–mRNA encoding the pore-forming subunit of the BKCa and of IKCa1–mRNAs (Figure Ic). In keeping with the observation that only functional expression of IKCa1 was altered after bFGF stimulation, the RT-PCR studies revealed a 3-fold increase in IKCa1 mRNA levels, whereas BKCa/Slo expression was unchanged (Figure Id).
The MEK/Extracellular Signal-Regulated Kinase Pathway Mediates Upregulation of IKCa1 Expression After Mitogenic Stimulation
Mitogen-induced activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK)–signaling system has been shown to upregulate IKCa1 expression and proliferation of fibroblasts and proliferating vascular smooth muscle cells in vitro.13,14 Therefore, we tested whether IKCa1 upregulation in HUVECs and HMEC-1 after stimulation with bFGF is mediated by activation of this signaling pathway. Pretreatment of HUVECs and HMEC-1 with the MEK inhibitor PD98059 (20 μmol/L) for 30 minutes before bFGF stimulation prevented the increase in IKCa1–mRNA (Figure Ib, Id, and Ie) and reduced IKCa1–mRNA levels below baseline observed in untreated controls (Figure Ib and Id). Consequently, the PD98059 treatment prevented the bFGF-induced increase of IKCa1 current amplitude in HUVECs (Figure Ie) and in HMEC-1 (Figure If). In contrast, the p38-mitogen-activated protein (MAP) kinase inhibitor SB203580 (5 μmol/L) and the c-jun N-terminal kinase (JNK) inhibitor SP600125 (20 μmol/L) had no effect on IKCa1 expression and current amplitudes after bFGF stimulation (data not shown). Inhibition of bFGF receptor tyrosine kinase by the tyrosine kinase inhibitor herbimycin A (1 μmol/L) or genistein (10 μmol/L) prevented bFGF-induced upregulation of IKCa1 in HMEC-1 (Figure Id). These results demonstrate the involvement of tyrosine kinase activity and activation of the MEK/ERK MAP kinase (MAPK) signaling pathway in bFGF-induced upregulation of IKCa1 in HUVECs and HMEC-1.
TRAM-34 and CLT Inhibit EC Proliferation After Stimulation With bFGF and VEGF
To elucidate whether the enhanced IKCa1 expression after mitogenic stimulation is functionally important, we tested whether the IKCa1 inhibitors TRAM-34, ChTx, and CLT suppress bFGF- or VEGF-stimulated mitogenesis of human ECs. Forty-eight hours after bFGF stimulation, the cell count (given as percent increase in cell count) of HMEC-1 and HUVECs increased to 194±7% (n=17) and 341±47% (n=16), respectively, whereas unstimulated HMEC-1 (99±4%; n=19; P<0.001 versus bFGF-stimulated cells) and HUVECs (107±6%; n=13; P<0.001 versus bFGF-stimulated cells) did not proliferate. After VEGF stimulation, the cell count of HMEC-1 and HUVECs increased to 169±37% (n=4) and 263±36% (n=13). TRAM-34 (IC50 5±1 nmol/L) and CLT (IC50 4±1 nmol/L) suppressed the bFGF-stimulated proliferation of HMEC-1 in a dose-dependent fashion, abolishing mitogenesis at 100 nmol/L CLT (P<0.01 versus bFGF alone; Figure 3A) or 100 nmol/L TRAM-34 (P<0.01 versus bFGF alone; Figure 3A). The inactive TRAM-7 (1 μmol/L) did not suppress proliferation (Figure 3A), indicating that the suppressive effect of TRAM-34 and CLT was not related to nonspecific toxicity. As shown in Figure 3B, mitogenesis in HUVECs was similarly blocked by 1 μmol/L TRAM-34 (P<0.01 versus bFGF alone) or 1 μmol/L CLT (P<0.01 versus bFGF alone) and by 100 nmol/L ChTx (P<0.01 versus bFGF alone).
Because HMEC-1 also express BKCa, we further tested whether the BKCa blockers IbTx and ChTx, which block BKCa and IKCa1, could suppress mitogenesis. IbTx (100 nmol/L) had no blocking effects (Figure 3A), whereas ChTx (50 nmol/L) abolished mitogenesis of HMEC-1 (111±3%; n=4; P<0.01 versus bFGF alone). This indicates that in contrast to IKCa1, BKCa is not essential for mitogenesis in these cells. The MEK inhibitor PD98059 (20 μmol/L) potently suppressed bFGF-induced mitogenesis in HMEC-1 (95±4%; n=18; P<0.001 versus bFGF alone) and in HUVECs (102±10%; P<0.01 versus bFGF alone), thus confirming that bFGF-induced mitogenesis is mediated via the Ras/Raf/MEK/ERK MAPK cascade.25 Together, these results suggest that upregulation of IKCa1 expression is required for EC proliferation after stimulation with angiogenic factor bFGF, as has been reported for mitogen-induced proliferation of lymphocytes, fibroblasts, and of vascular smooth muscle cells.12,14,15
TRAM-34 Suppresses Angiogenesis In Vivo
The mitogen-induced IKCa1 upregulation in HMEC-1 and in HUVECs and the effectiveness of the selective IKCa1 blocker TRAM-34 in suppressing bFGF-induced proliferation of HMEC-1 cells suggest that in vivo, IKCa1 blockade might reduce angiogenesis. We tested this hypothesis by using the Matrigel plug assay and administered TRAM-34 (120 mg/kg per day IP)15 or vehicle after introduction of the plug and daily thereafter for 2 weeks. Data and representative cross-sections of plugs from these experiments are shown in Figure 4. Although clear vascularization of plugs (presence of functional capillaries containing erythrocytes) was observed in all plugs of vehicle-treated mice, only few functional capillaries were present in the border region of plugs from 2 TRAM-34–treated mice and none in the other 5. Thus, vascularization (given as mean number of capillaries per section) was substantially suppressed by ≈85% (Figure 4). The 2-week TRAM-34 treatment caused no visible side effects or macroscopic organ damage. Together, this result suggests that in vivo blockade of IKCa1 reduces vascularization in this in vivo angiogenesis model.
Angiogenic factors such as bFGF and VEGF stimulate EC proliferation in vitro and angiogenesis in vivo. Using HUVECs and HMEC-1 as a model system, we demonstrated that stimulation by bFGF and VEGF upregulated endothelial expression of the IKCa1 gene and IKCa1 current amplitude and that selective IKCa1 blockade suppressed EC mitogenesis in vitro and angiogenesis in vivo, suggesting that IKCa1 channels play an important role in EC proliferation after stimulation by angiogenic factors.
Upregulation of IKCa1 expression has been reported similarly to contribute to the proliferation of growth factor–stimulated fibroblasts,14 vascular smooth muscle cells in vitro,15,16 neointimal vascular smooth muscle cells in situ,15 mitogen-activated human T lymphocytes, and cancer cells.9,11–13,17,26 In HUVECs and HMEC-1, like in fibroblast and vascular smooth muscle cells of the rat, increase of IKCa1 mRNA is mediated through the MEK/ERK–signaling cascade, whereas in T lymphocytes, augmentation of IKCa1 levels occurs as a result of activator protein 1 (AP1)-dependent transcription. Therefore, enhanced IKCa1 expression might be a functional characteristic of mitogen-activated and proliferating cells.9,14–17,26 Moreover, it is tempting to speculate that the enhanced expression and function of IKCa1, which has been detected in mesenteric endothelium of colonic cancer patients in situ,18 might be a consequence of increased serum levels of angiogenic factors often found in these patients.27
IKCa1 channels may promote EC mitogenesis and thus angiogenesis by enhancing the electrochemical driving force for Ca2+ influx via membrane hyperpolarization, which sustains the high-intracellular Ca2+ concentration required for gene transcription and cell cycle progression, as has been reported in lymphocytes, fibroblasts, and cancer cells.9,12,14,17,26 Although BKCa has also been proposed to be important in EC mitogenesis,8 the IKCa1 in HMEC-1 appears to play a more important role than BKCa in EC mitogenesis and in modulating Ca2+ signals of proliferating ECs. This can be explained by its higher Ca2+ affinity, which results in channel activation and thus membrane hyperpolarization at much lower intracellular Ca2+ concentrations.12,13,16,18,28–30 Induction of IKCa1 mRNA expression might thus be a required step for EC proliferation after stimulation with angiogenic factors.
Regarding the involvement of IKCa1 in the regulation of EC proliferation and angiogenesis, previous studies have shown that the fairly selective IKCa1 blocker CLT suppressed EC proliferation in vitro as well as angiogenesis in vivo.31 However, the precise mechanism by which CLT inhibits cell proliferation and angiogenesis is unclear because in addition to its blocking action on IKCa1, CLT inhibits cytochrome P450 epoxygenases.12 Noteworthy, the latter have also been suggested to be involved in mediating vascular homeostasis and angiogenesis.32 In the present study, we show that the highly specific IKCa1 inhibitor TRAM-34, which does not inhibit cytochrome P450 enzymes,12 was equally effective to inhibit EC proliferation. Moreover, in an in vivo model of angiogenesis, daily administration of TRAM-34 substantially reduced vascularization of Matrigel plugs without causing macroscopic side effects. In our hands, this demonstrates that the IKCa1 plays a crucial role in EC proliferation in vitro and seems to be important also in EC proliferation during angiogenesis in vivo.
In conclusion, upregulation of IKCa1 by angiogenic factors might be essential for promoting EC proliferation. Consistently, IKCa1 blockade by CLT and the highly specific IKCa1 inhibitor TRAM-34 suppresses EC proliferation, and IKCa1 blockade by TRAM-34 suppresses angiogenesis in vivo. In contrast to CLT, which is considered of limited clinical usefulness because of its severe liver toxicity,33 the highly selective IKCa1 blocker TRAM-34 may therefore have therapeutic value for preventing endothelial proliferation during tumor angiogenesis.
This work was supported by the Deutsche Forschungsgemeinschaft (FOR 341/5, 341/7, and 341/10; Ho 1103/2-4, GRK 276/2, and GRK 865/1). The authors thank Heike Wulff for the gift of TRAM-34 and TRAM-7 and for helpful suggestions on this manuscript.
I.G. and I.E. contributed equally to this work.
- Received April 21, 2004.
- Accepted December 28, 2004.
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