Published online before print June 8, 2006, doi: 10.1161/01.ATV.0000231518.86795.0f
© 2006 American Heart Association, Inc.
Vascular Biology |
VEGF Activates Receptor-Operated Cation Channels in Human Microvascular Endothelial Cells
From the Microvascular Research Laboratories (H.W.-C., R.R.F., D.O.B.) and Cardiovascular Research Laboratories (H.W.-C., A.F.J., J.C.H.), Department of Physiology, University of Bristol, UK.
Correspondence to Dr David Bates, Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, University of Bristol, Southwell St, Bristol BS2 8EJ, UK. E-mail Dave.Bates{at}bristol.ac.uk; or Prof Jules Hancox, Cardiovascular Research Laboratories and Department of Physiology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK. E-mail Jules.Hancox@bristol.ac.uk
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Abstract |
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Objective— Vascular endothelial growth factor (VEGF) exerts many of its effects by stimulating endothelial calcium influx, but little is known about channels mediating VEGF-induced cation entry. The aim of this study was to measure and characterize for the first time the VEGF-activated cation current in human microvascular endothelial cells (HMVECs).
Methods and Results— Whole-cell patch-clamp recordings were made from HMVECs. During applied voltage ramps, VEGF activated a current that reversed at 0 mV, was sensitive to gadolinium, and required extracellular cations. Noise analysis yielded a single-channel conductance of 27 pS. The current was not dependent on intracellular calcium stores, and was not blocked by inositol triphosphate (IP3) receptor or serine/threonine kinase inhibition but was partially inhibited by flufenamic acid. A similar current was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG). To determine whether VEGF could activate recombinant ion channels with similar properties, we investigated the effect of VEGF on Chinese hamster ovary cells cotransfected with VEGFR2 and the canonical transient receptor potential (TRPC) channels, TRPC3 or TRPC6. VEGF induced a similar current to that described above in VEGFR2-TRPC3 and VEGFR2-TRPC6 cells but not in cells transfected with either cDNA alone.
Conclusions— VEGF activates a receptor-operated cation current in HMVECs and OAG can activate directly a similar current in these cells. VEGF is also able to activate heterologously expressed TRPC3/6 channels through VEGFR2.
In human microvascular endothelial cells, VEGF activated a 27 pS, gadolinium-sensitive cation current, reversing at 0 mV, that was independent of calcium stores, IP3 receptors, or serine/threonine kinases. A similar current was activated by OAG and, in VEGFR2-TRPC3– and VEGFR2-TRPC6–transfected cells, by VEGF. VEGF activates a receptor-operated cation channel.
Key Words: VEGF • vascular permeability • ROC • endothelium
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Introduction |
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Vascular endothelial growth factor A (VEGF) is the primary angiogenic molecule in vertebrates in both physiological and pathological states. VEGF activation of its cognate receptor VEGFR2 (a receptor tyrosine kinase, also known as flk-1 or KDR1) on microvessel endothelial cells, the primary physiological mediator of VEGF actions, results in a complex signaling cascade2 that causes an interrelated and coordinated sequence of events leading to increased vascular permeability,3,4 breakdown of the extracellular matrix, and migration and proliferation of endothelial cells, resulting in new blood vessel formation.5 One of the earliest intracellular signaling events described for VEGF was an increase in intracellular calcium concentration ([Ca2+]i) of endothelial cells.6,7 This is required for most of the downstream functional changes induced by VEGF, including increased vascular permeability,8 angiogenesis,9 and vasodilatation10: the triumvirate of VEGF actions that result in increased nutrient delivery to tissue.
Many studies have measured the increase in [Ca2+]i in endothelial cells after VEGF treatment, described the time course of the response, the effects of inhibitors, and the downstream signaling of VEGFR2.6,7,11–14 Phospholipase C
(PLC),2 is recruited to Tyr1175, resulting in activation of the enzyme, generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).15 As Ca2+ entry plays a critical role in VEGF actions on microvessels, it is surprising, therefore, that nothing is known about the cation channels underlying VEGF-induced Ca2+ entry into the endothelial cells of microvessels.
In situ investigation of the microvasculature in vivo has provided evidence for a role in the permeability response of a PLC- and DAG-dependent, but protein kinase C (PKC)-independent, pathway for VEGF-mediated Ca2+ entry.16 In addition, the effect of VEGF can be mimicked by use of the membrane-permeant analog of DAG, 1-oleoyl-2-acetyl-sn-glycerol (OAG).17 Canonical transient receptor potential (TRPC) channels, TRPC3, TRPC6, and TRPC7 are a subgroup of the TRPC nonselective cation channels18 that can be directly stimulated by OAG19 and represent a pathway for receptor-operated Ca2+ entry. As TRPC3 and -C6 have been described in microvascular endothelial cells,20 it has been suggested that the DAG-activated TRPC channels, TRPC3 and TRPC6, participate in mediating the Ca2+ entry–dependent modulation of microvessel permeability by VEGF.17
At present, however, electrophysiological evidence for activation of cation channels by VEGF is lacking, and there are no published data regarding VEGF-activated cation currents in microvascular endothelial cells. Indeed, we have found only 4 electrophysiological studies to date that have used human microvascular endothelial cells (HMVECs).21–24 Therefore, taking an electrophysiological approach, the aims of the present study were (1) to record VEGF-induced cation influx into HMVECs, (2) to describe some of the properties of any such cation influx, and (3) to determine whether VEGF is able to activate candidate cation channels such as TRPC3 and TRPC6 channels. Primary human microvascular endothelial cells were used, firstly, as data arising would be of direct relevance to the human and, secondly, because VEGF acts to increase permeability and angiogenesis in microvascular endothelium, not in large vessels.
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Materials and Methods |
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Human microvascular endothelial cells were purchased from Cambrex (CC-2516). These are Clonetics human neonatal microvascular endothelial primary cultures from foreskin, which are assessed for endothelial specificity before shipping (positive for von Willebrand factor [vWF] and acetylated low-density lipoprotein and negative for smooth muscle actin) and have been shown to be positive for CD31, vascular cell adhesion molecule (VCAM), VE-cadherin, angiotensin-converting enzyme, and vimentin (see the online data supplement, available at http://atvb.ahajournals.org, for the relevant Cambrex Web-site address). On culture, the cells tested positive for VEGFR2 expression by Western blotting. The cells were cultured in EGM-2 MV media formulated by Cambrex for endothelial-specific growth on glass coverslips coated with attachment factor (Cambrex, Wokingham, Berks, UK). For details regarding the Chinese hamster ovary (CHO) cell transfection, see the online data supplement.
Electrophysiological measurements were made using whole-cell patch clamp (see the online data supplement for details). Once in the recording chamber, cells were bathed in a solution containing (in mmol/L): 140 NaCl, 5 CsCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH). The pipette dialysate contained (in mmol/L): 110 cesium methanesulfonate, 25 CsCl, 2 MgCl2, 3.62 CaCl2, 10 EGTA, 30 HEPES (pH adjusted to 7.2 with CsOH) with a calculated free [Ca2+] of 100 nmol/L. The EGTA in the pipette was included to buffer bulk intracellular calcium, such that calcium-dependent currents would not be activated. For IP3 receptor inhibition studies (Results and Figure 2), 5 mg/mL heparin was included in the pipette dialysate, so that it could access the cell interior by intracellular dialysis after attaining the whole-cell recording configuration. Once the whole-cell configuration had been obtained, the external superfusate was rapidly switched to a solution containing 50 µmol/L Ca2+. A voltage step from the holding potential of –60 mV to –100 mV for 50 ms, followed by an ascending voltage ramp from –100 mV to +100mV (dV/dt, 0.5 V sec–1; holding potential of –60 mV) was used to measure whole-cell membrane current. The protocol was repeated every 5 seconds. For experiments using external Na+-free and Ca2+-free solution, N-methyl-D-glucamine (NMDG) was substituted for external Na+; no substitution for external Ca2+ was made. The following chemicals were added to the superfusion solution singly or in combination as required: 1 nmol/L VEGF, 100 µmol/L OAG, 100 µmol/L Gd3+, 200 nmol/L staurosporine (SS), 100 µmol/L flufenamic acid (FFA).
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Spectral Analysis of Current Variance
The variance of the VEGF and OAG-activated currents was calculated from the integral of spectral density function, as described elsewhere.25,26 The VEGF- or OAG-sensitive spectra were fitted by a double Lorentzian function (see the online data supplement for details).
Statistics
For statistical tests used, see the online data supplement. A value of P<0.05 was considered significant.
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Results |
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VEGF-Mediated Currents in HMVECs
To determine whether VEGF induced a current in HMVECs, voltage ramps were applied before and during VEGF exposure. To eliminate activation of calcium-dependent cation entry attributable to release of calcium from intracellular stores, these experiments were carried out in the presence of EGTA in the pipette to buffer bulk intracellular [Ca2+]. VEGF induced a sustained inward current at a negative holding potential of –60 mV (Figure 1A) and increased both the inward and outward current during applied voltage ramps (Figure 1B). From 21 cells, we measured a VEGF-induced current of –1.58±0.4 pA/pF at –60 mV. The current reversed at –1.85±1.1 mV (not significantly different from 0 mV) and was outwardly rectifying at positive voltages (Figure 1B and 1C). Superfusion with 100 µmol/L Gd3+ (Figure 1D and 1F) abolished the current (–0.14±0.04 pA/pF at –60 mV holding potential, n=10, P<0.05 compared with VEGF; Figure 2E). The inward current component was inhibited (Figure 1E and 1F) by replacement of extracellular cations with NMDG (0.11±0.08 pA/pF at –60 mV holding potential, n=5, P<0.05 compared with VEGF; Figure 2E), showing that the current was a cation current.
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VEGF induced an augmentation in current noise associated with an increase in the inward current at the holding potential (Figure 1G). Examination of the power spectra at frequencies greater than 0.98 Hz in the absence and presence of VEGF shows that the power of the noise was increased by VEGF (Figure 1H). This increased noise arises from channel gating and, itself, provides valuable information concerning the ion channels underlying the VEGF-activated current.24,25 Because the open channel conductance is a function of the variance of the current, a power spectral analysis of the currents yields an estimate of the single-channel conductance.25 The power spectrum was fitted with a double Lorentzian (see the online data supplement for details), the corner frequencies of which (fc1 and fc2) are functions of the gating kinetics of the underlying channel and the asymptotes at low frequency, S(0)1 and S(0)2, are functions of the power of the noise resulting from channel gating.25 The power spectrum of the VEGF-activated current had mean S(0)1=5.99±3.74 pA2s and S(0)2=0.32±0.29 pA2s and mean fc1=2.20±0.64 Hz and fc2=48±9 Hz (n=3). Assuming that the VEGF-activated current represented a single population of nonselective cation channels,26 the mean single-channel conductance was 26.6±8.9 pS (n=3).
Ligand-dependent cation currents are generally mediated through either store-operated or receptor-operated cation channels. Store-operated cation channels are dependent on IP3 receptor activation, which is blocked by heparin. To determine whether IP3 receptor activation was required for the VEGF-mediated current, recordings were made with intracellular dialysis of heparin from the patch pipette (5 mg/mL of pipette solution). This did not affect either the size of the VEGF-induced current at –60 mV holding potential (–1.3±0.4 pA/pF, P>0.1 compared with VEGF) or the shape of the current voltage relationship (Figure 2A), ruling out a role under these conditions for IP3-mediated activation of cation entry.
Receptor-operated cation channels are often activated by DAG, either directly, or through PKC activation. To determine whether the VEGF-activated current was activated by PKC or other serine/threonine kinases, the effect was determined of the wide-ranging serine/threonine kinase inhibitor staurosporine. The VEGF-mediated current was not affected by staurosporine (–1.37±0.44 pA/pF at –60 mV holding potential, P>0.1 compared with VEGF; Figure 2B). The mean densities of VEGF-activated current at –60 mV alone and in the presence of Gd3+, cation removal, staurosporine, and heparin are shown in Figure 2E. To verify further that this was not a PKC-dependent current, recordings were made in the presence of 1 µmol/L of the PKC activator phorbol myristate acetate. This did not result in any increase in current in 4 cells (Figure I in the online data supplement), although VEGF was still able to induce a current following phorbol myristate acetate treatment. Collectively, these observations indicate that the VEGF-activated cation current was not contingent on PKC mobilization.
Flufenamic acid has been shown to affect differentially 2 receptor-operated cation channels, TRPC3 and TRPC6. Although FFA is a wide-ranging inhibitor of ion channels, blocking chloride and large conductance potassium channels, it has been shown to discriminate between TRPC3, which it inhibits, and TRPC6, which it stimulates.27 To investigate the possible contribution of these TRPC subunits to the VEGF-mediated current, the effect of FFA was examined. In approximately half of the cells, the VEGF-mediated current was partially inhibited by FFA (eg, Figure 2C and 2D), the remainder of the cells being unaffected by FFA (Figure 2D). Although VEGF alone increased the current density by 2.4±0.3 fold compared with control, FFA significantly reduced the VEGF-mediated current by 0.81±0.08-fold (P<0.05 compared with 1).
HMVECs Display an OAG-Sensitive Current
To determine whether or not HMVECs could respond to a membrane-permeant analog of DAG, 100 µmol/L of OAG was applied. A sustained inward current was induced by OAG with a similar time course of onset and magnitude as that induced by VEGF over the 240 seconds that measurements of both responses were made (black trace in Figure 3A). The current at –60 mV averaged –0.70±0.24 pA/pF (Figure 3C), reversed at –0.14±0.94 mV, was outwardly rectifying at positive voltages, and was inhibited by Gd3+ (Figure 3B), and the inward, but not the outward, component of the current was abolished by replacement of extracellular cations with NMDG (Figure 3B and 3C). This current was similar to the VEGF-activated current described above. Figure 3D shows the noise of the holding current before (black) and after (gray) addition of OAG; the inset shows the original current traces. The power spectrum for the OAG-activated current (Figure 3E) was fitted by a double Lorentzian function with mean±SE S(0)1=2.55±0.70 pA2s, S(0)2=0.17±0.07 pA2s and mean fc1=7.8±2.1 Hz and fc2=96±17 Hz (n=5). Assuming that the OAG-activated current represented a single population of nonselective cation channels, the mean single-channel conductance was 23.2±8.5 pS (n=5).
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VEGF Activation of VEGFR2 Can Activate Recombinant TRPC3 and TRPC6 Channels
As highlighted in the introduction, TRPC3 and -C6 can be directly stimulated by DAG/DAG analogs,19 have been reported to be present in microvascular endothelial cells,20 and have been proposed to participate in mediating the Ca2+ entry–dependent modulation of intact microvessel permeability by VEGF.17 We confirmed the presence of TRPC3 and TRPC6 protein in the HMVECs used for the present study with immunofluorescence and Western blotting (supplemental Figure II). Direct activation of TRPC3/C6 channel currents by VEGF has not hitherto been demonstrated and therefore we performed additional experiments on recombinant TRPC3 and -C6 channels, in which 1 nmol/L VEGF was applied to CHO cells cotransfected with VEGFR2 and TRPC3 (R2-C3) or TRPC6 (R2-C6). This resulted in an increase in inward holding current at –60 mV and increased both the inward and outward current during applied voltage ramps (Figure 4). Reversal potentials for both TRPC channel–transfected cells lay near 0 mV (mean±SEM –1.63±1.24 mV for R2-C3 and –0.2±1.2 mV for R2-C6). Current–voltage relationships showed an outwardly rectifying outward current at voltages greater than 0 and an inward current at negative voltages for both R2-C3 (Figure 4A through 4C) and R2-C6 (Figure 4D through 4F). This current was not seen in VEGFR2, TRPC3, or TRPC6 singly transfected cells or in nontransfected cells in response to VEGF (Figure 4C and 4F). Figure 4G shows the mean±SEM effects of VEGF treatment of R2-C3 and R2-C6 current densities. We determined that the VEGF-induced current in VEGFR2/TRPC-transfected cells had similar properties to that induced in HMVECs, by examining the effect of extracellular cation removal and Gd3+/FFA application (supplemental Figure III).
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Discussion |
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Despite numerous studies that have measured the effect of VEGF on calcium concentrations in microvascular endothelial cells by imaging or ratiometric measurement using fluorescent dyes,11,13,14,17,28 the properties of the cation currents that may be responsible for the VEGF-induced increase in [Ca2+]i have not hitherto been established. In particular, it has not been ascertained as to whether these currents were receptor-operated or store-operated cation currents, although many reviews have assumed that they are store operated because VEGF activates PLC
and, hence, IP3 production,29 and some downstream effects in large artery endothelial cells (eg, prostacyclin production) are inhibited by chelating intracellular calcium.30 We describe here, for the first time, the characteristics of a nonselective cation current activated by VEGF in microvascular endothelial cells. This significantly extends the existing information on the electrophysiological properties of human microvascular endothelial cells (for which there is comparatively little such information,21–24 possibly because these cells are difficult to record from). This report also provides the first evidence that VEGF activates a receptor-operated cation channel current in human microvascular endothelial cells that is store, IP3 receptor, and PKC independent. Significantly, the DAG analog, OAG, induced similar currents in these cells. Because the channels activated by VEGF are cation permeable, they are likely to contribute to Ca2+ entry, either directly as Ca2+ entry pathways themselves or indirectly through Na+ entry and modulation of Na+/Ca2+ exchange.31 This study also provides the first direct demonstration of the activation by VEGF of a cation current through recombinant TRPC3 and -C6 channels in heterologous expression systems. Although our data do not demonstrate unequivocally that the VEGF-activated cation current in HMVECs was carried by TRPC3/C6, they are concordant with the possibility that VEGF could act through these channel types.
VEGF Activates a Receptor-Operated Cation Current
The principle that tyrosine kinase receptors can act through receptor-operated channels has been established in other systems, as these channels have been implicated in platelet-derived growth factor (PDGF) stimulation of vascular smooth muscle cells32 and brain-derived neurotrophic factor (BDNF) activation of TrkB receptor in neurons.33 However, the observations in this study constitute the first evidence that VEGF increases cation entry into human microvascular endothelial cells through activation of a current with electrophysiological properties matching those of receptor-operated channels. Significantly, the VEGF-induced current was present in the absence of store release, during IP3 inhibition, and was mediated by ligand activation.
The Identity of the Receptor-Operated Cation Channel
On the basis of the data in this study, it is not possible to attribute conclusively a specific molecular identity to the channels underlying the VEGF-activated cation currents in HMVECs. Although it might be argued that channel knockdown of particular TRPC subtypes would provide insight into their role in the VEGF response of HMVECs that we have observed, neither siRNA or antisense knockdown of ion channels has previously been demonstrated in microvascular endothelial cells, let alone followed by successful patching and electrophysiological recording. Moreover, it is worth noting that gene knockdown strategies cannot necessarily be assumed to resolve unambiguously the role of particular TRPC types in responses seen in native tissues because of the potential for both false-positive and false-negative results.34
It is possible, however, to make qualified inferences regarding the properties of the channels, on the basis of data obtained in this study. Thus, the characteristics of the measured whole-cell currents and the single-channel conductance of 27 pS reported here are similar to those previously reported for 2 receptor-operated cation currents, TRPC619 and TRPC3. TRPC6 has a single-channel conductance of 25 to 35 pS, reverses at 0, is inhibited by Gd3+, gated by OAG, independent of IP3 activation, and not sensitive to PKC inhibition, features concordant with the observations made from HMVECs here. Moreover, the parameters of the noise spectrum of the TRPC6 current of rabbit portal vein smooth muscle cells25,26 are strikingly similar to those of the VEGF- and OAG-activated currents in the present study. TRPC3 is also inhibited by Gd3+, reverses at 0, and gated by OAG19 but has a dual conductive state (65 and 17 pS35). Thus, the value of 27 pS obtained in the present study, being intermediate between 17 and 65 pS, does not exclude the involvement of a TRPC3 channel in the VEGF-activated current. The single-channel conductance of these conductive states cannot be resolved by analysis of the power spectrum of the whole-cell currents as performed in this study.
Although TRPC6, TRPC3, and TRPC7 have all been shown to gate OAG-induced calcium influx, Gd3+ has previously been shown to inhibit TRPC3 and TRPC6. However, Gd3+ does not affect TRPC7 channels.36 This precludes a contribution of TRPC7 to the OAG and VEGF-activated currents seen in HMVECs in this study but is consistent with involvement of TRPC3 or -C6. Although TRPC3 has also been associated with IP3 receptor activation,35,37 the results shown here indicate that inhibition of IP3 receptor activation does not inhibit the VEGF-mediated response. However, PLC activation in IP3 receptor–deficient cell lines38 still activates TRPC3. Therefore, the involvement of TRPC3 cannot be ruled out.
Considered collectively, the electrophysiological data from HMVECs in this study are concordant with TRPC3 and -C6 as prime molecular candidates for the channels underlying the observed responses to VEGF. Furthermore, the presence in the HMVECs used in this study of proteins for TRPC3 and -C6 was verified by immunofluorescence and Western blotting (see supplemental Figure II), whereas the data from VEGFR2 and TRPC3 or -C6 cotransfected cells provide "proof of concept" that these TRPC channel subtypes can be activated by VEGF. On the basis of these findings, future work using knockdown of TRPC3 and -C6 proteins may be warranted if these strategies can be successfully applied to HMVECs and if potential limitations with the techniques34 are taken into account when interpreting the findings that emerge.
Some comparison is warranted between our findings and those of Jho et al,12 who have recently suggested the involvement of TRPC1 in the VEGF-activated Ca2+ entry in human (macrovascular) umbilical vein endothelial cells (HUVECs). Neither the single-channel conductance (27 pS) nor the reversal potential (
0 mV) of the VEGF-activated current in the present study is consistent with the involvement of TRPC1 channels in the HMVEC current responses to VEGF. TRPC1 channels would be expected to have a single-channel conductance of <10 pS.39 Moreover, TRPC1 channel currents, in the presence of extracellular calcium, and with cesium as the dominant intracellular ion have a reversal potential of +23 mV.40 Differences between the results of the present study and those of Jho et al may be explained by functional differences between the Ca2+ signaling kinetics and mechanisms in intact endothelial cells of microvessels and of large vessel endothelial cells in culture. For example, in measurements of Ca2+ in intact microvessels (frog mesentery and rat venules), VEGF induced a transient increase in Ca2+ that returns to control levels,14,17,28 whereas in large vessel endothelial cells in culture (HUVEC), VEGF induces a biphasic response, an increase followed by a plateau level, characteristic of store-mediated Ca2+ entry.6,11,13 Furthermore, previous studies using thapsigargin to deplete stores showed that VEGF could still increase calcium, implicating Ca2+ entry through TRPC3 and TRPC6.28 This study did not show, however, that TRPC1 was not able to contribute to the increase in calcium entry induced by VEGF in the absence of store depletion. Thus, although TRPC1 is unlikely to have contributed to VEGF-activated currents in HMVECs in this study, and our observations are consistent with previous observations made from intact microvessels in situ, our findings do not preclude a role for TRPC1 in mediating endothelial responses to VEGF in other parts of the vasculature.
In summary, this study demonstrates clearly that VEGF induces a receptor-operated cation channel current in human microvascular endothelial cells. This current has characteristics similar to those of VEGF-mediated TRPC currents in cells heterologously expressing VEGFR2 and TRPC3 or TRPC6. These data therefore constitute the first evidence of VEGF-mediated currents in human microvascular endothelial cells. Although the molecular identity of the underlying channels remains to be determined, the characteristics of the data reported here are consistent with the notion that calcium entry stimulated by VEGF involves nonselective cation entry through TRPC-like channels.
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Acknowledgments |
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Sources of Funding
This work was supported by the British Heart Foundation grants PG02/082 (to D.O.B. and J.C.H. for H.-W.C.) and BB2000003 (to D.O.B.).
Disclosure(s)
None.
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Footnotes |
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Original received December 15, 2005; final version accepted May 18, 2006.
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