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Signaling pathways mediating VEGF165-induced calcium transients and membrane depolarization in human endothelial cells Nancy S. Dawson,* David C. Zawieja,*1 Mack H. Wu,† and Harris J. Granger* *Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, Texas A&M University System Health Science Center, College Station, Texas, USA; and †Department of Surgery, U.C. Davis Medical Center, M.I.N.D. Institute, Sacramento, California, USA To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3923fje SPECIFIC AIMS Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and microvascular permeability and accelerates tumor growth and metastases. The aims of the present study were 1) to simultaneously image changes in intracellular calcium concentration ([Ca2⫹]i) and membrane potential in VEGF165 –stimulated human umbilical endothelial cells (HUVEC), and 2) to determine how specific signal transduction molecules and ion channels may modulate these transients.

utilized the organic channel blockers, LOE-908 (which inhibits NSCC1 and NSCC2) and SKF96365 (which inhibits both store-operated calcium channels (SOC) and NSCC2). LOE-908 reduces the calcium peak to 35– 45% of the control, abolishes the calcium plateau, and blocks depolarization by ⬃50 – 60% (Fig. 1C); SKF shows a similar calcium profile and blocks depolarization by ⬃80% (Fig. 1D). 2. Inhibitors of VEGFR2-receptor tyrosine kinase, src kinase, and inositol-triphosphate (IP3) eliminate both the calcium and membrane potential transients

PRINCIPAL FINDINGS 1. VEGF165 induces rapid changes in cytosolic calcium concentration and membrane potential in HUVEC. Changes in intracellular calcium and membrane potential were monitored simultaneously in HUVEC using the fluorescent indicators, indo-1 AM and DiSBAC2(3), respectively. Application of VEGF165 (100 ng/ml) induces a rapid increase in [Ca2⫹]i), followed by a sustained plateau phase where [Ca2⫹]i, remains slightly elevated above baseline (Fig. 1A). Membrane potential also shows a biphasic response. Initially, as [Ca2⫹]i increases, a slight, transient membrane hyperpolarization occurs, but as the [Ca2⫹]i peaks and then decays toward control, a strong depolarization develops, which is sustained (Fig. 1A). Increases in [Ca2⫹]i may reflect release from internal calcium stores and/or influx from the extracellular space. We determined the relative contribution from each calcium compartment and evaluated the concomitant effect on membrane polarization. Pretreatment with thapsigargin (TG) depletes calcium stores so that no calcium transients are elicited by VEGF165, and depolarization is diminished to ⬃50%. Blockade of nonselective calcium entry channels (NSCC) with LaCl3 results in ⬃40% inhibition of the calcium peak and abolishes the plateau. To further elucidate the role of specific calcium entry channels, we 0892-6638/06/0020-0991 © FASEB

Because many proangiogenic characteristics of VEGF are mediated through VEGFR2, specific inhibitors of four known molecules in the VEGF165-VEGFR2 signal transduction cascade [ras, phospholipase C␥l (PLC ␥l)-inositol phosphate (IP3), src kinase and phosphatidylinositol-3-kinase (PI3K)] were used to probe their involvement. We found that calcium and membrane potential transients are both eliminated with inhibitors to VEGFR2 receptor tyrosine kinase (Fig. 1B), src kinase (PP2), and the IP3 receptor (IP3R) (2-APB). These data strongly suggest the sufficiency and necessity of VEGFR2 and the downstream requirement for src kinase and IP3-operated calcium channels to elicit the calcium and membrane potential transients. However, in the 10-min period of VEGF stimulation studied, we did not see evidence of immediate downstream ras signaling, since a farnesyltransferase inhibitor (FTI277) (that blocks ras translocation to the plasma membrane), did not alter [Ca2⫹]i or membrane potential. 1

Correspondence: Lymphatic Biology Division, Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114 USA. E-mail: [email protected] doi: 10.1096/fj.05-3923fje 991

explored the involvement of specific chloride channels. Pretreatment of HUVEC with the voltage-independent, chloride channel blocker, tamoxifen gave a similar profile as that observed with wortmannin. CONCLUSIONS AND SIGNIFICANCE To our knowledge, this is the first study to use calciumand membrane-sensitive fluorescent dyes to investigate their dynamic and concomitant relationship in VEGF165-stimulated endothelial cells, and to use inhibitors to identify key signaling molecules that modulate these transients. Binding of VEGF165 to VEGFR2 initiates a complex signaling pathway, known to involve dimerization and phosphorylation of the tyrosine kinase receptor, recruitment of src-homology (SH)-2 domain-containing proteins, (PI3K and PLC␥1), elevation of [Ca2⫹]i, and calcium influx. Calcium response

Figure 1. A) Simultaneous responses of cytosolic calcium and membrane potential in HUVEC following application of 100 ng/ml human recombinant VEGF165. The time of addition of VEGF165 is indicated by the vertical line. For comparison, control responses to VEGF165 obtained from Figure 1A are represented by a dotted line without error bars in panels B–D. B) Effect of VEGFR-2 inhibition on calcium transients and membrane depolarization after application of 100 ng/ml VEGF165. C) Effect of pretreatment of HUVEC with LOE-908, the nonselective cation channel blocker (NSCC1 and NSCC2) on calcium and membrane potential responses to VEGF165. D) Effect of pretreatment of HUVEC with SKF96365, the store-operated and nonselective cation channel blocker (SOC and NSCC2), on calcium and membrane potential responses to VEGF165.

3. Inhibitors of PI3 kinase and chloride channels modulate membrane depolarization, in a calciumindependent manner PI3K is known to directly associate with ligated VEGFR2. To test its involvement in the rise of [Ca2⫹]i and the shift in membrane potential, we used a PI3Kspecific inhibitor, wortmannin, and found that it reduces membrane depolarization by ⬃60% but does not significantly alter the calcium transient. Because calcium currents are believed to be too small to explain the significant depolarization that we observed in VEGF165-activated cells (see Fig. 1B), we 992

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We show that VEGFR-2 activation significantly increases [Ca2⫹]i and shifts the resting membrane potential in HUVEC. A rise in [Ca2⫹]i can reflect release of Ca2⫹ from intracellular stores, entry of Ca2⫹ from the extracellular space, and the concerted effect of several calcium-sequestration mechanisms. We show that VEGF165 induces a biphasic increase in [Ca2⫹]i, consisting of an initial strong calcium peak that lasts for ⬃2 min, followed by a sustained plateau phase that remains above baseline for the 10-min period of observation (Fig. 1A). By using various inhibitors of calcium influx pathways, we show that the strong calcium peak represents a combination of both internal calcium stores release and calcium influx, since blockade with LaCl3, LOE-908, and SKF96365 all produce a reduction in the initial calcium peak. Depletion of calcium stores with TG or inhibition of stores release with 2-APB, completely blocks both the initial peak and the plateau phase of the calcium transient. Calcium influx is believed to occur through capacitative calcium channels (CCE)/SOC and/or receptoractivated NSCC channels. Our experiments with LaCl3, LOE-908, SKF 96365 and 2-APB substantiate this entry pathway for calcium influx, and implicate SOC-like channels as the primary calcium influx pathway for refilling stores in VEGF165-stimulated cells. Inhibitors of VEGFR-2 and the IP3R abolish stores release and calcium influx (Fig. 1B), an inhibitor of src kinase abolishes calcium stores release, whereas, inhibitors of PI3K and chloride channels do not significantly alter stores release or calcium influx. VEGF165-stimulation of VEGFR2 tyrosine kinase activity is necessary for calcium signaling (Fig. 1B). The studies using 2-APB show that stores release and calcium influx require IP3/IP3R signaling presumably through IP3R-gated channels in the endoplasmic reticulum, near the plasma membrane. The studies with PP2 suggest a dominant role for src kinase, a SH-2 domain-containing protein, in regulating internal calcium release, possibly through activating the PLC␥1-IP3-IP3R cascade.

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The calcium transients implicate calcium entry, activated by stores-depletion in a manner that may open chloride channels, such as calcium-activated chloride channels (CaCC) and perhaps vol-regulated anion channels (VRAC). Such channels are thought to be involved in maintaining a polarized membrane and stabilizing the driving force for calcium influx. We show that inhibitors of chloride channels and PI3K do not directly alter the calcium transients but strongly influence the membrane potential. Membrane depolarization Potassium channels (including Ca2⫹-activated K⫹ channels, KCa, inward-rectifying K⫹ channels, Kir, and voltage-dependent K⫹ channels) are the major class of ion channels thought to determine membrane potential. Cultured, preconfluent HUVEC cells, like those used in our experiments, are reported to have a resting membrane potential of ⬃⫺27 to ⫺52 mV. Additionally, chloride channels are also implicated as regulators of membrane potential in endothelial cells. Endothelial cells with more negative potentials are termed K⫹-type cells (expressing a more prominent hyperpolarization via potassium channels) and cells with less negative potentials are called Cl⫺ type cells (expressing the involvement of chloride channels). Activation of VRAC and CaCC cause depolarization and are suggested to be modulators of the inward driving force for calcium, and NSCC are also proposed to “tune” the membrane potential in resting and activated cells. VEGF165 stimulation clearly shifts the resting membrane potential of HUVEC (Fig. 1A). Immediately after stimulation, a slight transient hyperpolarization develops, which is followed by a strong and sustained depolarization. The biphasic nature of the membrane potential profile is a “mirror-like image” of the concomitant, biphasic changes occurring in the calcium transients (Fig. 1A) (i.e., where the slight hyperpolarization appears to be correlated with calcium release (peak) and the pronounced depolarization phase correlated with the sustained phase of calcium influx). We found that various inhibitors of calcium influx (Fig. 1C and D) attenuate the membrane depolarization 50 – 80%, showing a definite calcium-influxsensitive component to membrane depolarization. Inhibitors of the calcium stores release and influx pathway (e.g., VEGFR2/src kinase-/PLC␥1-IP3-IP3R) abolish the membrane depolarization completely, whereas inhibitors of PI3K and chloride channels attenuate the membrane depolarization by ⬃50%. We propose that KCa are activated in response to the rise in [Ca2⫹]i, accounting for the initial membrane hyperpolarization, which provides a favorable electrochemical gradient for calcium entry and enhances the elevation of intracellular [Ca2⫹]i (peak). Depletion of calcium stores activates calcium entry through SOC channels, which shift the membrane potential toward depolarization, thereby stabilizing the driving force for calcium

VEGF-INDUCED CALCIUM AND DEPOLARIZATION IN HUVEC

Figure 2. Schematic depiction of the integrated response of the endothelium to VEGF stimulation, showing key signaling molecules that appear to regulate calcium and membrane potential transients.

influx, and providing negative feedback for additional calcium entry. The precise mechanism for this is not understood; however, the experiments with LaCl3, SKF96365, PP2, 2-APB, and RTKI clearly show that a large rise in [Ca2⫹]i is strongly coupled to the depolarization. The prolonged depolarization may activate anion channels in the plasma membrane, such as CaCC and/or VRAC/ (I Cl, swell), or potentially by KCa . Several experiments strongly suggest that some aspects of membrane depolarization are calcium-dependent and others are calcium-independent. Since calcium influx inhibitors (LOE 908 and SKF96365) attenuated the membrane depolarization, calcium influx may be responsible for the calcium-dependent portion of membrane depolarization. VEGF-mediated calcium influx is abolished, and the magnitude of depolarization is significantly reduced when internal calcium stores are depleted (TG or LaCl3). TG pretreatment (30 min) completely abolished the VEGF-induced calcium transients but only partially attenuated depolarization, suggesting a potential role for calcium-independent activation of ion channels such as NSCC, VRAC, or closure of Kir channels. The wortmannin and tamoxifen profiles for calcium and membrane potential also suggest that VEGF-induced membrane depolarization is regulated, in a similar manner. We propose that the PI3 kinase pathway may directly or indirectly activate VRAC, as we observed a partial depolarization from VEGF165 after pretreatment with wortmannin, without alteration of the VEGF-induced calcium transients. In conclusion, we show that VEGF165 alters [Ca2⫹]i and membrane potential in HUVEC cells. VEGF induces a rapid peak in calcium followed by a sustained plateau above baseline, and these calcium changes are accompanied by a transient hyperpolarization and a sustained large depolarization, respectively. Figure 2 depicts our understanding of the integrated response of the endothelium to VEGF stimulation, and shows key signaling molecules that appear to regulate calcium and membrane potential transients.

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Signaling pathways mediating VEGF165-induced calcium transients and membrane depolarization in human endothelial cells Nancy S. Dawson,* David C. Zawieja,*,1 Mack H. Wu,† and Harris J. Granger* *Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, Texas A&M University System Health Science Center, College Station, Texas, USA; and †Department of Surgery, U.C. Davis Medical Center, M.I.N.D. Institute, Sacramento, California, USA Cytosolic calcium and membrane potential were monitored simultaneously in quiescent human umbilical vein endothelial cells (HUVEC) exposed to vascular endothelial growth factor (VEGF)165 using the fluorescent indicators indo-1 AM and DiSBAC2(3), respectively. Application of VEGF165 to cells elicits a rapid rise in cytosolic calcium followed by a slower decline toward control values. Peak calcium is associated with a slight membrane hyperpolarization; however, as calcium falls toward control, a strong depolarization develops and is sustained throughout a 10-min period of VEGF165 stimulation. Both the VEGF165mediated rise in cytosolic calcium and membrane depolarization are eliminated by inhibitors of VEGFR-2, tyrosine kinase, src kinase and inositol-1,4,5 triphosphate-operated calcium channels. Calcium entry, which is initially facilitated by transient hyperpolarization, is restricted by a substantial, sustained depolarization that developed during the downstroke of the calcium spike. Inhibition of plasmalemmal calcium channels diminished the magnitude and duration of the calcium spike, suggesting that extracellular calcium influx, secondary to stores release, is a significant component of the calcium transient. Inhibition of chloride channels substantially reduced membrane depolarization. In addition, the depolarization is modulated by PI3 kinase in a ras-independent manner. In summary, intracellular calcium and membrane potential are influenced by several key signaling cascades of VEGFR-2 activation in HUVEC.—Dawson, N. S., Zawieja, D. C., Wu, M. H., Granger, H. J. Signaling pathways mediating VEGF165induced calcium transients and membrane depolarization in human endothelial cells. FASEB J. 20, E141–E149 (2006) ABSTRACT

Key Words: endothelium 䡠 membrane potential 䡠 intracellular calcium 䡠 signal transduction

Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis (1, 2) and microvascular permeability (3, 4). The protein is produced by a wide variety of tissues, but its actions are relatively specific to vascular endothelial cells (5). At least three 0892-6638/06/0020-0141 © FASEB

different VEGF-binding surface receptors have been identified (6). However, many of the actions of VEGF, including mitogenesis (7, 8) and vascular hyperpermeability (9, 10), are mediated primarily by the VEGFR2/flk-1/KDR receptor. Ligation of VEGFR-2 by VEGF elicits at least four parallel signaling cascades originating from the activation of ras, phospholipase C␥1 (PLC␥1), src kinase, and phosphatidylinositol-3-kinase (PI3 kinase) (11–14). VEGF-activated endothelial cells demonstrate a transient increase in cytosolic calcium (9, 15–17). This event is crucial to the final physiological responses set in motion by binding of the ligand to its receptor. Some studies point to intracellular stores as the initiator of the calcium transient (18), whereas others emphasize the importance of calcium influx from the extracellular fluid (15, 17). Although the electromotive driving force is an important determinant of the rate of calcium entry into endothelial cells (19 –21), the impact of VEGFR-2 activation by VEGF on membrane potential is unknown. The aims of the present study were threefold: 1) to elucidate the role of each of the four major signaling cascades activated by VEGFR-2 ligation in mediating VEGF165-induced changes in calcium and membrane potential in human endothelial cells, 2) to determine the effect of VEGF165 on changes in membrane potential and explore the role of specific ion channels and 3) to estimate the relative importance of calcium entry and intracellular calcium release in the generation of the calcium transient elicited by VEGF165. To achieve these objectives, cytosolic calcium and membrane potential were monitored simultaneously in confluent monolayers of human umbilical vein endothelial cells using fluorescent indicators. Inhibitors of different specific signaling elements and ion channels were used to evaluate their contributions to the overall regulation 1

Correspondence: Lymphatic Biology Division, Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA. E-mail: [email protected] doi: 10.1096/fj.05–3923fje E141

of intracellular calcium and membrane potential by VEGF165.

MATERIALS AND METHODS Cell culture Human umbilical vein endothelial cells (HUVEC) were obtained from Cambrex (#CC-2519, Walkersville, MD) as passage one and were grown in endothelial growth medium (EGM) (Cambrex, Walkersville, MD) supplemented with 10% FBS (HyClone, Logan, UT). For the experiments, typically, passage three cells were used and grown to ⬃80 –95% confluence on 1.5% gelatin-coated, round-glass coverslips in 10% FBS-supplemented EGM. The cells were then “quiesced” in a step-down fashion before the experiment by placing them initially in 2% FBS-supplemented EGM (4 – 8 h) and finally overnight in endothelial basal media (EBM) (Cambrex, Walkersville, MD) supplemented with 0.5% FBS, 50 U/ml heparin, GA-1000, hydrocortisone, and 0.1% BSA (USB, Cleveland, OH). Simultaneous measurement of cytosolic calcium and membrane potential Fluorescent measurements of cytosolic calcium and membrane potential were measured simultaneously using cells loaded with both the calcium-sensitive dye, indo-1 AM and the membrane potential-sensitive dye, DiSBAC2(3) (22). HUVEC, which had been grown on coverslips, were washed twice with an indo-loading buffer (5.4 mM KCl, 136.8 mM NaCl, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 0.3 mM Na2HPO4, 11.1 mM D-glucose, 5.5 mM HEPES, 2.0 mM CaCl2, and 0.1% BSA, pH 7.4) and then loaded for 1 h. at 37°C with 2 ␮M indo-1 AM (Molecular Probes, Eugene, OR) in the indoloading buffer. Cells were then washed twice in a scanning buffer (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM D-glucose, 15 mM HEPES, 0.1% BSA, pH 7.4) and loaded for 30 min at 37°C with 100 nM DiSBAC2(3) (Molecular Probes, Eugene, OR) in the scanning buffer. Agonist or inhibitor reagents were typically preincubated with the cells during the 30 min DiSBAC2(3) loading step. The cells were not washed before observation because a constant external source of the indicator in the external medium is required for estimating membrane potential. A suitable field of view containing 10 to 25 cells was found using phase optics. The cells were imaged using a Zeiss ⫻40 C-Apochromat water-immersion lens (numerical aperture (NA) 1.2) on an Ultima-Z laser confocal microscope system (Meridian Instruments, Okemos, MI). The loaded cells were excited simultaneously with UV (354 nM) and green laser lines (405 nM) for indo-1 AM and DiSBAC2(3), respectively. The cells were maintained in the scanning buffer at 37°C with a stage warmer and scanned every 20 s over a 10- to 20-min period. Agonist solutions were added in 20-␮l volumes and mixed using a continuous, low-velocity microstirrer. Spectra were collected simultaneously at the dual-emission wavelengths for indo-1 AM (405 and 485 nM) and the single emission wavelength for DiSBAC2(3) (580 nM), using the appropriate bandpass filters to create three separate images for each field of view at each time point. Background fluorescence at each of the three wavelengths was measured and subtracted from the appropriate image. Individual regions of interest were defined, and raw fluorescence intensity was measured. Wavelength crossover compensation between the two dyes was performed based on the results of separate E142

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single-dye loading studies. From the background-subtracted and crossover-compensated values, the 405/485 nm ratio value was used as a direct index of intracellular calcium (i.e., an increase in the ratio indicates an increase in calcium), and the 580 nm value was used as a relative index of membrane potential (i.e., an increase in the corrected 580 nm signal indicates a relative depolarization in membrane potential). The values of intracellular calcium and membrane potential in the individual cells within each experimental region were then averaged and represented as an n of 1. Experimental treatments were repeated in separate plates of cells 5 to 19 times for each group. Both the calcium and membrane potential data were normalized to the values measured in the first scan of the control period. The data were then synchronized to the time at which VEGF was added, which typically occurred 5– 6 min after control data were obtained, and then plotted as normalized data vs. time. Agonists and inhibitors The primary agonist, human recombinant VEGF165 (Peprotech, Rocky Hill, NC), was added real-time as a single dose, during scanning, at 100 ng/ml final concentration, to establish the control VEGF-induced calcium- and membrane potential responses. Effects of other agonists and inhibitors on the VEGF-induced responses were evaluated by preincubating the cells for 30 min with these reagents during the DiSBAC2(3) loading step (see above). The following is a list of the drugs and the final concentrations used: 2-APB, 75 ␮M (Sigma-Aldrich, St. Louis, MO); clomiphene, 10 ␮M (SigmaAldrich); FTI-277, 200 ␮M (Calbiochem, San Diego, CA); LaCl3, 300 ␮M (Sigma-Aldrich); LOE-908, 100 ␮M (gift from Boehringer-Ingelheim, Ridgefield, CT); LY294002, 50 ␮⌴ (Biomol, Plymouth Meeting, PA); LY83583, 10 ␮M (BioMol); PP2, 10 ␮M (Calbiochem); PP3, 10 ␮M (Calbiochem); SKF96365, 50 ␮M (Calbiochem): tamoxifen, 4-hydroxy-(Z), 10 ␮M (Calbiochem); thapsigargin, 1␮M (Calbiochem); (Z)3-[(2,4-dimethyl-3-(ethoxycarbonyl) pyrrol-5-yl)methylidenyl]indolin-2-one, a specific VEGFR-2 receptor kinase inhibitor (RTKI), 10 ␮M (Calbiochem); and wortmannin, 250 nM (BioMol). Statistics The values of intracellular calcium and membrane potential in the individual cells (10 –25 cells per microscopic field) on each coverslip were calculated, averaged, and represented as an n of 1. Experimental treatments were repeated in separate plates of cells (one treatment per plate) 5 to 19 times for each treatment. Statistical differences were determined by ANOVA, post hoc Fisher’s least significant difference (LSD), and Student’s t tests and were considered significant at P ⬍ 0.05.

RESULTS Figure 1A demonstrates the responses of cytosolic calcium and membrane potential in HUVEC to VEGF. Application of VEGF to a confluent monolayer of HUVEC leads to a calcium spike that peaks in less than 2 min, followed by a decline to a plateau value slightly higher but significantly different from the control concentration. A transient hyperpolarization occurs during the calcium spike, but the membrane depolarizes relative to resting voltage as intracellular calcium

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Figure 1. A) Simultaneous responses of cytosolic calcium and membrane potential in human umbilical endothelial cells (HUVEC) following application of 100 ng/ml human recombinant vascular endothelial growth factor vascular endothelial growth factor165 (VEGF165). The time of addition of VEGF165 is indicated by the dashed vertical line. For comparison, control responses to vascular endothelial growth factor165 obtained from Figure 1A are represented by a dotted line without error bars in panels B–D. B) Effect of VEGFR-2 inhibition (RTKI) on calcium transients and membrane depolarization after application of 100 ng/ml vascular endothelial growth factor165. C) Effect of pretreatment of HUVEC with LOE-908, the nonselective cation channel blocker (NSCC1 and NSCC2) on calcium and membrane potential responses to VEGF165. D) Effect of pretreatment of HUVEC with SKF96365, the store-operated and nonselective cation channel blocker (SOC and NSCC2) on calcium and membrane potential responses to VEGF165.

falls from its peak value. We have documented similar transients of these two variables in HUVEC monolayers exposed to histamine and thrombin, agonists known to activate the phosphoinositide signaling cascade through phospholipase ␤1 (data not shown). The typical cytosolic calcium and membrane potential responses to VEGF, shown in Figure 1A, were used for comparison and designated as the control response (n⫽19) in all subsequent figures. Numerous studies support the primacy of the VEGFR-2 receptor in mediating the mitogenic and VEGF-INDUCED CALCIUM AND DEPOLARIZATION IN HUVEC

hyperpermeability properties of VEGF. To determine whether the calcium and membrane potential transients are dependent on this specific receptor, the VEGFR-2-specific receptor kinase inhibitor RTKI was applied to HUVEC before stimulation with VEGF. As shown in Figure 1B, both the calcium and membrane potential transients were eliminated by pretreatment with the VEGFR-2-specific kinase inhibitor. Thus, VEGFR-2 activation is a key factor in the initiation of VEGF-induced changes in cytosolic calcium and membrane polarization in HUVEC. From a theoretical viewpoint, entry of extracellular calcium can contribute to the calcium peak, as well as account for the steady-state elevation of cytosolic calcium above basal levels after activation with VEGF. To address this issue, we examined the effect of pretreatment with the calcium entry blockers LOE-908 and SKF 96365 on the calcium transient (Figure 1, C and D, respectively). LOE-908 and SKF 96365 eliminated the steady-state increment in cytosolic calcium to VEGF stimulation normally observed at the end of the calcium transient and reduced the calcium peak to 35– 45% of the control response. Inhibition of calcium influx reduced the VEGF-elicited membrane depolarization by ⬃75%. A similar result was obtained by pretreatment of HUVEC with LaCl3 (Figure 2A), another blocker of nonspecific calcium channels. Pretreatment with thapsigargin, a blocker of calcium reuptake into the endoplasmic reticulum, depletes internal stores and reduces the calcium and membrane potential responses to subsequent addition of VEGF (Figure 2B). The importance of store-emptying in the response to VEGF is supported by the similar effect of thapsigargin per se (Figure 2C) and VEGF on calcium and membrane potential transients in HUVEC. Src kinase is one of the four major enzymes that binds to, and is activated by, the cytoplasmic domain of VEGFR-2. The src kinase inhibitor PP2 completely eliminates the VEGF-induced transients in cytosolic calcium and membrane potential (Figure 3A). By contrast, the inactive analog PP3 had no effect (Figure 2D). Recent studies (23) suggest that src kinase is an important component of VEGFR-2 activation of the phosphoinositide signaling cascade. Phospholipase C␥1 (PLC␥1) is activated by ligation of VEGFR-2 (11). Activation of PLC␥1 by VEGF leads to the production of diacylglycerol (DAG) and inositol 1,4,5 triphosphate (IP3), an important regulator of calcium channels in the endoplasmic reticulum membrane (18, 24). DAG participates in cell signaling mainly through activation of classical and novel isoforms of protein kinase C. In preliminary studies, we found no effect of pretreatment with the protein kinase C (PKC) inhibitors 19 –27 peptide and bisindolylmaleimide I (data not shown). IP3 opens ligand-operated channels in the endoplasmic reticulum (ER), leading to emptying of internal calcium stores into the cytosol (25) and may contribute to extracellular calcium influx through IP3 receptor channels in the plasma membrane as well (26, 27). Figure 3B demonstrates that the E143

chloride channels to this component of the membrane potential pattern (30 –32). Pretreatment of HUVEC with the chloride channel blockers tamoxifen (Figure 3D) and clomiphene (Figure 4C) eliminated or substantially reduced the magnitude of depolarization. The calcium responses in the control and chloride channel inhibitor groups were not statistically different. VEGFR-2 ligation leads to association of adaptor proteins with the cytoplasmic domain of the receptor; these adaptor proteins, in turn, serve as guanine nucleotide exchange factors that promote the conversion of inactive ras-guanidine diphosphate (ras-GDP) to active ras-guanidine triphosphate (ras-GTP) (33). Ras-GTP plays a key role in stimulating the p42/44 mitogen-

Figure 2. A) Effect of pretreatment with LaCl3. Responses after pretreatment are depicted by solid lines with error bars. Control responses to VEGF165 are represented by the dotted line without error bars. The dashed vertical line indicates the time of application of VEGF in the control and experimental group. B) Effect of pretreatment with thapsigargin before addition of VEGF. C) Effect of thapsigargin per se on calcium and membrane potential in HUVEC. The dashed vertical line in this panel indicates the time of application of thapsigargin or vascular endothelial growth factor. D) Effect of pretreatment of HUVEC with PP3, an inactive form of the src inhibitor PP2, on calcium and membrane potential responses elicited by VEGF165

IP3 receptor blocker 2-APB eliminates the calcium and membrane potential transients normally elicited by VEGF, supporting a central role of IP3 in VEGF signaling. The transient hyperpolarization elicited by exposure of HUVEC to VEGF is probably due to opening of calcium-activated potassium channels, as shown previously in studies of endothelial cell electrophysiology (28, 29). In the present study, our major focus was on the events responsible for the sustained membrane depolarization that follows the transient hyperpolarization. Because some believe that calcium currents are too small to explain depolarizations in endothelial cells (29), we examined the possible contribution of E144

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Figure 3. A) Effect of pretreatment of HUVEC with src kinase inhibitor PP2 on calcium and membrane potential changes elicited by VEGF165. Responses after pretreatment are depicted by solid lines with error bars. Control responses to VEGF165 are represented by the dotted lines without error bars. The dashed vertical line indicates the time of application of VEGF in the control and experimental groups. B) Effect of pretreatment of HUVEC with 2-APB, an inhibitor of inositol 1,4,5 triphosphate (IP3) binding to calcium-release channels in the endoplasmic reticulum. C) Effect of pretreatment of HUVEC with wortmannin, a phosphatidylinositol-3kinase (PI3) kinase inhibitor. D) Effect of pretreatment of HUVEC with an inhibitor of chloride channels, tamoxifen.

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altered. Because wortmannin inhibits myosin light chain kinase at the doses used in this study, we performed the same experiments with LY294002, another putative PI3 kinase inhibitor that does not have MLCK inhibitory actions at the concentrations used here. LY294002 (Figure 4B) had the same effect as wortmannin on calcium and membrane potential transients elicited by VEGF165. Thus, PI3 kinase appears to play a substantial role in modulating ion channels responsible for the sustained depolarization elicited by VEGFR-2 activation. Cyclic nucleotide gated (CNG) channels are nonspecific cation channels that are activated by store-operated calcium entry (29, 34). On emptying of the internal calcium store, the resting nonselectivity of the channel with respect to Na⫹ and K⫹ is altered to favor Na⫹ entry, thus leading to membrane depolarization. Cyclic GMP activates cyclic nucleotide gated channels. Since VEGFR-2 activation leads to NO production (23, 35, 36) and elevated levels of cGMP (35, 37), we pretreated HUVEC with LY83583, a putative inhibitor of guanylate cyclase and thus CNG channel activity, before exposure to VEGF (Figure 4D) This inhibitor reduced the plateau of the steady-state membrane potential response to VEGF by an average of 25%, but this effect was not statistically significant. The peak calcium concentration was elevated by 14%, possibly reflecting a role for cyclic GMP in activation of calcium extrusion from the cytosol (38).

DISCUSSION Figure 4. A) Effect of pretreatment of HUVEC with farnesyltransferase inhibitor FTI-277 on calcium and membrane potential responses elicited by VEGF165. Responses after pretreatment are depicted by solid lines with error bars. Control responses to VEGF165 are represented by the dotted line without error bars. The dashed vertical line indicates the time of application of VEGF in the control and experimental group. B) Pretreatment of HUVEC with LY294002, a specific inhibitor of phosphotidylinositol-3-kinase. C) Pretreatment with clomiphene, a chloride channel blocker. D) Pretreatment with LY83583, an inhibitor of guanylate cyclase and CNG channels.

activated protein kinase (p42/44MAP) kinase signaling pathway. The participation of the small G protein in the receptor tyrosine kinase signaling is dependent on membrane-bound ras. Farnesylation of ras is required to direct the G-protein to the plasmalemmal membrane. Pretreatment with the farnesyltransferase inhibitor (FTI-277) has no effect on the calcium transient or membrane potential responses following VEGF stimulation (Figure 4A). PI3 kinase is the fourth major signaling molecule that directly associates with ligated VEGFR-2 (11, 14). As shown in Figure 3C, pretreatment of HUVEC with wortmannin, a PI3 kinase inhibitor, reduces the membrane depolarization elicited by VEGF by nearly 60%. By contrast, the calcium transient was not significantly VEGF-INDUCED CALCIUM AND DEPOLARIZATION IN HUVEC

The present study focuses on the mechanisms underlying calcium and membrane potential transients elicited by VEGF in quiescent, confluent monolayers of human endothelial cells. To our knowledge, this is the first analysis of VEGF signaling based on simultaneous measurements of cytosolic calcium and membrane potential. Although several studies have described the effect of VEGF on cytoplasmic calcium in endothelial cells (9, 17, 18, 24, 39), we are not aware of previous measurements of VEGF-induced changes in membrane potential. In this study, the cytoplasmic calcium signal reaches a peak value ⬃100 s after exposure of HUVEC to VEGF. During the upstroke of the calcium signal, membrane potential exhibits a transient hyperpolarization that reverses to depolarization as intracellular calcium concentration declines toward the control value. The magnitude of the sustained depolarization is nearly five times greater than the transient hyperpolarization. At the end of ten minutes exposure to VEGF (the plateau phase), the cytosolic calcium signal is slightly, but significantly, elevated above the control concentration and the membrane potential signal is ⬃45% higher than control. Using simultaneous recordings of fluorescence intensity of probes specific for cytosolic calcium and membrane potential, we examined the impact of specific inhibitors of specific proE145

teins to gain greater insight into VEGF signaling in HUVEC. VEGFR-2 is required for initiation of calcium and membrane potential responses to VEGF165 The signaling pathways activated by vascular hyperpermeability factor/vascular endothelial growth factor have received extensive attention. In the vascular endothelium, the peptide is capable of binding to the extracellular domain of two of the three known receptor tyrosine kinases, namely VEGFR-1 and VEGFR-2. VEGFR-1 has a much higher affinity for VEGF165 (KD of 0.02 vs. 0.8 nM for VEGFR-2) (40). With some exceptions (41), most studies point to a dominant role for the lower affinity VEGFR-2 in mediating endothelial cell proliferation and vascular hyperpermeability. This view is supported not only by dose-response relationships, but also by studies using specific antireceptor blocking antibodies (9, 16), receptor-specific peptide agonists (7) and kinase-specific inhibitors (8, 10). In the present study, both the calcium and membrane potential transients were eliminated by a specific VEGFR-2 tyrosine kinase inhibitor, thus providing further support for the primacy of the lower affinity receptor in VEGF signaling in HUVEC. Src-kinase and IP3 are required for initiation of calcium and membrane potential responses to VEGF165 Ligation of the VEGFR-2 receptor leads to receptor dimerization, autophosphorylation of specific tyrosine residues on their cytoplasmic tails and subsequent association of signaling proteins with these src homology SH2-binding domains (5, 11). Phospholipase C␥1 is one of several SH2-containing proteins capable of binding to VEGFR-2 after receptor autophosphorylation (23). Docking of phospholipase C␥1 (PLC␥1) to the cytoplasmic tail of VEGFR-2 activates tyrosine phosphorylation and leads to increased production of IP3 (24). IP3 mediates the emptying of intracellular calcium stores through opening of IP3-operated calciumrelease channels located in the endoplasmic reticulum (25). IP3 and its breakdown products may also act on ion channels in the plasma membrane (26, 27, 42). The importance of IP3 in causation of the calcium and membrane potential responses is evidenced by the ability of the IP3-receptor blocker, 2-APB, to prevent these responses on exposure of cells to VEGF. Recent studies indicate that 2-APB may also inhibit plasmalemmal channels capable of conducting calcium after store depletion (43). However, since the initiating event (e.g., store release) is prevented by 2-APB, the effect of the inhibitor on calcium influx is secondary and does not alter our conclusions. Additional indirect evidence that the emptying of intracellular stores is an important component in the VEGF signaling paradigm is provided by the patterns of calcium and membrane depolarization that are elicited by treatment of HUVEC E146

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with thapsigargin, which blocks calcium reuptake into the endoplasmic reticulum and stimulates the release of endoplasmic reticular calcium stores (Figure 2C). Stimulation of HUVEC with thapsigargin produced initial patterns of changes in calcium and membrane depolarization similar to those seen during VEGF treatment. Src kinase is usually viewed as a second parallel signaling pathway set in motion by docking of the enzyme to the cytoplasmic SH2-domains generated by VEGFR-2 autophosphorylation. However, some studies support the notion that src kinase activity is required for full activation of PLC␥1. This leads to a series arrangement of VEGFR-2, src and PLC␥1 (23), where the tyrosine phosphorylation of PLC␥1 after VEGF exposure is reduced by the src kinase inhibitor, PP2. Our results are consistent with this view since pretreatment with PP2 eliminated both the calcium and membrane potential responses normally elicited by VEGF while the inactive analog PP3 had no effect. The critical role of the VEGFR-2/src kinase/PLC␥1/IP3 axis in governing endothelial calcium and membrane potential responses to VEGF is highlighted by the fact that only inhibitors of VEGFR-2 tyrosine kinase, src kinase and the IP3-receptor eliminated both the calcium and membrane potential responses. An important pathway activated by VEGFR-2 ligation is the ras-MAP kinase cascade. Pretreatment of HUVEC with a farnesyltransferase inhibitor had no effect on calcium and membrane potential responses in HUVEC. This is consistent with our previous findings that activation of the p42/44 MAP kinase pathway by VEGF in HUVEC is relatively ras-independent and mainly due to stimulation of eNOS with subsequent phosphorylation of RAF-1 by cGMP-dependent protein kinase (44). Calcium and chloride channels contribute to the calcium and membrane potential responses to VEGF165 Ion channels in the plasma membrane of endothelial cells play a role in VEGF signaling by influencing the rate of entry, and the instantaneous concentration of calcium ions. These effects can be further categorized into changes in the electrochemical driving force and in the conductivity of calcium-conducting channels. In endothelial cells, changes in the membrane potential reflect the combined, relative contributions of potassium, nonselective cation and chloride channels (29). The transient hyperpolarization observed immediately after VEGF stimulation is probably due to activation of calcium-dependent potassium (KCa) channels that open during the upstroke of cytosolic calcium concentration (28). The resting membrane potential of HUVEC is generally more negative than the equilibrium potential for chloride (29). Thus, opening of volume-regulated (VRAC) and calcium-activated (CACC) chloride channels should cause depolarization. In our studies, the chloride-channel blockers clomiphene and tamoxifen substantially depressed the

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steady-state depolarization normally elicited by VEGF. A similar reduction in the membrane potential response was produced by inhibition of calcium entry with LaCl3, LOE-908, and SKF96365 or alternatively by inhibition of PI3 kinase with wortmannin and LY294002. Thus, the membrane depolarization elicited by VEGF appears to exhibit calcium-dependent and calcium-independent components. The effect of PI3 kinase is not likely due to subsequent Akt activation of endothelial nitric oxide synthase (eNOS) and the associated production of cGMP, as the soluble guanylate cyclase inhibitor LY83583 did not lead to a statistically significant depression of the membrane potential response to VEGF. Because of their low conductances, plasmalemmal calcium channels do not play a major role in modulating membrane potential (29). Their importance lies in their ability to conduct calcium from the extracellular fluid into the cytosol, thereby regulating the concentration of free cytosolic calcium ions and downstream calcium-dependent effectors such as eNOS, ion channels, and other proteins. On the other hand, cytosolic calcium concentration at any given time is dependent on the balance or imbalance between calcium entry, release from intracellular stores, and removal of the cation from the cytosol by pumps and exchangers. Previous studies in endothelial cells demonstrate the existence of a store-operated plasmalemmal calcium channel (SOC) that is activated on emptying of sequestered calcium pools (44 – 46). Moreover, calcium can enter through nonspecific cation channels (NSCC1 and NSCC2) capable of conducting calcium ions (34, 45). To further elucidate the role of specific calcium entry channels, we used the organic channel blockers, LOE-908 (which inhibits NSCC1 and NSCC2) and SKF96365 (which inhibits both SOC and NSCC2). In addition, NSCC1 and NSCC2 were blocked with exposure to LaCl3. In our studies, inhibition of calcium entry with LOE-908, SKF96365, and LaCl3 substantially reduced the calcium peak, implying that calcium flow through plasmalemmal ion channels is important in generating the transient calcium spike. The calcium spike is effectively eliminated with an IP3 inhibitor (2-APB), which may prevent both stores release and plasmalemmal SOC-mediated calcium influx, thereby eliminating membrane depolarization altogether. In addition, the inhibitors of calcium entry through SOC, NSCC1, and NSCC2 eliminated the sustained elevation of cytosolic calcium concentration in the plateau phase, providing further testimony to the action of these agents on calcium influx from the cell exterior. Moreover, suppression of calcium entry severely depresses membrane depolarization, suggesting that channels responsible for VEGF-induced membrane depolarization (e.g., nonspecific cation and chloride channels) have a strong calcium-dependent nature. On the other hand, the depolarization elicited by opening of these channels may play a significant role in turning off the calcium switch through a reduction in the electromotive force driving calcium into the cell. A more detailed VEGF-INDUCED CALCIUM AND DEPOLARIZATION IN HUVEC

analysis of the contribution of various ion channels to VEGF signaling will require direct measurements of channel activity using patch clamping and other electrophysiological techniques. As discussed above, pretreatment with wortmannin and LY294002, inhibitors of PI3 kinase, produced VEGF-elicited responses very similar to that observed after blockade of the chloride channels. Others have demonstrated that PI3-kinase mediates opening of chloride channels directly (46) or via a rho-dependent action (47). In addition, PI3 kinase can increase the conductance of nonspecific cation channels (48), providing an additional mechanism for generating a sustained depolarization after VEGF stimulation of human endothelial cells.

Physiological significance of calcium and membrane potential responses elicited by VEGF165 in endothelial cells The calcium spike elicited by VEGF in endothelial cells is a key event in the initiation of vasodilation, hyperpermeability, and angiogenesis at the microvascular concentration. In microvessels (9, 15) and cultured cells (16, 24, the current study), the peak of the calcium transient occurs in the time range of several seconds to 2 min. To the extent that membrane potential is a key determinant of calcium influx in endothelial cells (20, 21), hyperpolarization enhances calcium entry at a time when plasmalemmal calcium conductance is at its height; depolarization secondary to opening of chloride channels contributes to turning the calcium switch off. Although eNOS activity can be enhanced by VEGFinduced activation of AKT and PKC, the primary stimulus for NO production is elevated cytosolic calcium. The VEGF-induced vasodilator signal is NO diffusing from the endothelial cells to underlying vascular smooth muscle and therefore exits the VEGF signaling cascade at eNOS. The hyperpermeability and mitogenic signals are conducted downstream through subsequent activation of the soluble guanylate cyclase/ protein kinase G axis, and terminate in RAF phosphorylation and activation of the p42/44 MAP kinase cascade (44). The activities of MEK1/2 and ERK1/2, downstream elements of the MAP kinase pathway, reach a peak in 2 to 5 min (49), a time course similar to VEGF–induced venular hyperpermeability to serum albumin. The mitogenic signal continues to ERK1/2, but the hyperpermeability signal appears to exit the MAP kinase cascade at MEK1/2 (50). An interesting aspect of the current study is that most of the mediators of cytosolic calcium and membrane potential (i.e., src, IP3, PI3 kinase) set in motion by VEGF165 reside close to the receptor, rather than in the more distant elements of the signaling pathway (i.e., guanylate cyclase/PKG axis or p42/44 MAP kinase cascade). In conclusion, we show that VEGF165 alters [Ca2⫹]i and membrane potential in HUVEC. VEGF induces a E147

rapid peak in calcium followed by a sustained plateau above baseline, and these calcium changes are accompanied by a transient hyperpolarization and a large sustained depolarization, respectively. To our knowledge, this is the first analysis of VEGF165 signaling based on concurrent measurements of cytosolic calcium and membrane potential. We believe these findings provide important insights into the key roles that calcium and membrane potential play in the VEGF165-activation of endothelial cells. The authors would like to thank Shelley Hohl and E. Lynn Wink for technical help with the statistical analysis and production of figures. This research was supported by grants HL-058062 (HJG) and HL-075199 (DCZ) from the NHLBI.

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