Orai1 and CRAC Channel Dependence of VEGF-Activated Ca2+ Entry and Endothelial Tube FormationNovelty and Significance
Rationale: Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to endothelial biology is mostly unknown.
Objective: The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation.
Methods and Results: In human umbilical vein endothelial cells, Orai1 disruption by short-interfering RNA or dominant-negative mutant Orai1 inhibited calcium release–activated (store-operated) calcium entry, VEGF-evoked calcium entry, cell migration, and in vitro tube formation. Expression of exogenous wild-type Orai1 rescued the tube formation. VEGF receptor-2 and Orai1 partially colocalized. Orai1 disruption also inhibited calcium entry and tube formation in endothelial progenitor cells from human blood. A known blocker of the immune cell CRAC channel (3-fluoropyridine-4-carboxylic acid (2′,5′-dimethoxybiphenyl-4-yl)amide) was a strong blocker of store-operated calcium entry in endothelial cells and inhibited calcium entry evoked by VEGF in 3 types of human endothelial cell. The compound lacked effect on VEGF-evoked calcium-release, STIM1 clustering, and 2 types of transient receptor potential channels, TRPC6 and TRPV4. Without effect on cell viability, the compound inhibited human endothelial cell migration and tube formation in vitro and suppressed angiogenesis in vivo in the chick chorioallantoic membrane. The compound showed 100-fold greater potency for endothelial compared with immune cell calcium entry.
Conclusions: The data suggest positive roles for Orai1 and CRAC channels in VEGF-evoked calcium entry and new opportunity for chemical modulation of angiogenesis.
Angiogenesis is an endothelial migration and tube formation process underlying new vessel growth from preexisting blood vessels.1 It has roles in wide-ranging physiological events and pathologies that include diabetic retinopathy, peripheral vascular disease, endometriosis, tissue regeneration, atherosclerosis, obesity, rheumatoid arthritis, and cancer.2,–,4 Strategies to enhance or suppress angiogenesis (depending on the clinical situation) have attracted much attention. In the case of efforts to stimulate angiogenesis for cardiovascular therapy, preclinical data have been encouraging, whereas late-stage clinical trials have been disappointing.5,6 Problems with the complexities of angiogenesis and the differing biology of old patients and young animal models have been suggested as explanations for the difficulties in translating to the clinic, suggesting that greater understanding and new intervention approaches are needed.6 Strategies aimed at inhibiting angiogenesis to reduce tumor expansion have, however, seen success, although with limitations, for example, resulting from drug resistance, again because of the complexities of the angiogenesis.3,7 Perhaps surprisingly, angiogenesis inhibition may have advantage in cardiovascular disease settings because it could be a means to suppress obesity and metabolic diseases that drive atherosclerosis.4
Primary intercellular signaling molecules in angiogenesis are the vascular endothelial growth factors (VEGFs), which bind to VEGF receptors such as VEGFR2.1,7 The receptors are tyrosine kinase receptors that link to downstream signaling pathways such as monomeric G proteins and phosphatidylinositol 3 and mitogen-activated protein kinases, but there is also evidence for associated intracellular Ca2+ signals originating from phospholipase Cγ and inositol 1,4,5-trisphosphate.1,8 These Ca2+ events show the classic Ca2+-release followed by Ca2+-entry characteristics.8,9 Jho et al9 suggested that TRPC1 (transient receptor potential canonical 1) channels contribute to the Ca2+-entry mechanism and other investigators have suggested roles of TRPC6 channels in angiogenesis.10,11 Synergism of VEGF and TRPC1 in zebrafish angiogenesis has been identified.12 Nevertheless, the underlying Ca2+ channel mechanisms and their significance remain incompletely understood.
A Ca2+ channel type with emerging prominence is the Ca2+-release activated Ca2+ (CRAC) channel.13,–,15 It is an extremely low-conductance, highly calcium-selective channel that opens in response to any signal that depletes Ca2+ in intracellular stores.14 A pore-forming subunit of the channels is Orai1, a member of the Orai family of tetraspanin-related membrane proteins.13,–,15 The channel type was discovered in immune cells and individuals with disrupted Orai1 function, because of a R91W mutation, have severe combined immune deficiency.13,16 With the identification of Orai1, however, it has become increasingly apparent that the CRAC channel is widely distributed and thus not restricted to the immune system. Its general activation mechanism through STIM1 of depleted Ca2+ stores14 is common to many cell types, such that most mammalian cells exhibit store-operated Ca2+ entry that may be accounted for, at least partly, by Orai1. The general activation signal of store-depletion suggests that it is a nodal point in the signaling of multiple agonists, presenting an opportunity for overcoming problems of redundancy, compensation, and drug resistance. Importantly, there is specific evidence that human umbilical vein endothelial cells (HUVECs) express Orai1, exhibit store-operated Ca2+ entry that depends on Orai1, and contain a small CRAC channel-like current.17 HUVECs are commonly used as a basis for in vitro angiogenesis assays. The relevance of CRAC channels and Orai1 to the VEGF responses of these, or other, endothelial cells is unknown.
Here, we investigated the relevance of Orai1 and CRAC channel-related Ca2+ entry to VEGF-evoked Ca2+ entry and endothelial cell behavior, using a variety of established angiogenesis assays.1 In addition to molecular manipulation of Orai1, which may not only relate to the CRAC channel, we investigated a chemical blocker that has been found to have selectivity for CRAC channels in immune cells.18,19 HUVECs, endothelial cells cultured from human saphenous vein obtained at coronary artery bypass operations, and late outgrowth endothelial progenitor cells (EPCs) from healthy volunteers were used in addition to chicken embryo chorioallantoic membrane for in vivo angiogenesis. EPCs may be important in vasculogenesis, vascular repair, and tumor vessel formation.3,20,21
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Cell Preparation and Culture
HUVECs were cultured in EGM-2 growth medium supplemented with EGM-2 bullet kit (Lonza) at 37°C in a humidified atmosphere containing 5% CO2. Provided in the Online Data Supplement are the methods for culture of EPCs, endothelial cells from human saphenous vein, HEK 293 cells with and without stable expression of TRPC6, and CHO cells stably expressing TRPV4 (transient receptor potential vanilloid 4).
Short Interfering RNAs and cDNAs
Cells at 90% confluence were transfected with 20 nmol/L short interfering (si)RNA using Lipofectamine 2000 in OptiMEM according to the instructions of the manufacturer (Invitrogen). Sequences of siRNA probes are given in the Online Data Supplement. Fresh EGM-2 growth medium was added after 4 to 6 hours and the cells were analyzed 48 hours after transfection. To validate effectiveness of siRNA probes, mRNA was isolated and quantified by real-time RT-PCR (see the Online Data Supplement). Orai1 siRNA 1 (Or1.si.1) effectiveness was also confirmed by Western blotting (see the Online Data Supplement). When cells were transfected with human Orai1 (accession no. BC013386) or its dominant negative (DN) mutant (R91W) in pcDNA6, 0.2 μg DNA was added. Orai1 cDNA was intended to saturate Or1.si.1 in rescue experiments. eYFP-STIM1 was in pDS (from J. Liou, Stanford University Medical School, CA).
Intracellular Ca2+ Measurement
Endothelial cells were incubated with fura-2AM for 1 hour at 37°C followed by a 0.5-hour wash at room temperature (21±2°C). Fluo-4AM was used in place of fura-2AM for recordings of overexpressed TRPC6 and TRPV4 activity. Measurements were made at room temperature on a 96-well plate reader (FlexStation, Molecular Devices). The change (Δ) in intracellular calcium (Ca2+) concentration was indicated as the ratio of fura-2 emission intensities for 340 nm and 380 nm excitation (F ratio). Wells within columns of the 96-well plate were loaded alternately for test and control conditions. The recording solution contained (mmol/L): 130 NaCl, 5 KCl, 8 d-glucose, 10 Hepes, 1.2 MgCl2, 1.5 CaCl2, titrated to pH 7.4 with NaOH. When Ca2+-free extracellular solution was used, CaCl2 was omitted; for Ca2+ add-back, Ca2+ was 0.2 mmol/L. S66 was added 15 minutes before Ca2+ measurements at room temperature.
Cells were labeled with antibodies using standard immunofluorescence protocols (details are given in the Online Data Supplement) and visualized using a deconvolution system (Applied Precision Instruments, Seattle, WA) based on the Olympus IX-70 inverted microscope with ×60 objective (NA 1.4).
Whole-Cell Patch-Clamp Recordings
See the Online Data Supplement.
Cell Migration and Viability
Cell migration assays were performed using a modified Boyden chamber (8-μm pores; BD Biosciences, UK). HUVECs transfected with siRNA or pretreated for 24 hours with S66 were serum starved in EGM-2 for 4 hours, resuspended at 5×104 cells/mL in EGM-2 containing 0.4% FCS, and loaded in the upper chamber. The lower chamber contained 0.4% FCS supplemented with the chemoattractant 20 ng/mL VEGF (Sigma). After incubation for 4 hours at 37°C in a 5% CO2 incubator, the inserts were fixed with 70% ethanol for 2 minutes. Cells on duplicate membranes were scraped from the upper surface, and migrating cells underneath were stained with hematoxylin/eosin and evaluated by counting cells in 6 randomly chosen fields under light microscopy. Methods for measuring cell viability are given in the Online Data Supplement.
In Vitro Tube Formation
HUVECs were detached by trypsinization and, after neutralization of trypsin, were counted and resuspended in EGM-2 containing 1% FCS and 2 ng/mL VEGF at 4×105 cells/mL. HUVECs were seeded at 2×104 cells per well in Matrigel-precoated BD Biosciences 96-well plates (catalog no. 354149). Tube formation was quantified 18 to 20 hours later. Cells were treated with chemical agents for 24 hours or transfected 48 hours before seeding on the 96-well plate. Chemical agents were maintained throughout experiments. EPCs were plated on thin-layer Matrigel precoated 24-well plates (BD Biosciences, catalog no. 354605) at 5×104 cells/well in 1% FCS EGM-2 with 2 ng/mL VEGF, and tube formation was quantified 20 hours later. Digital images of endothelial tubes were obtained by bright-field light microscopy. Tube formations were measured blind by an independent observer, giving: (1) total tube length per image; and (2) number of complete loops (circles) per image.
In Vivo Angiogenesis
Chicken embryo chorioallantoic membrane angiogenesis assays were performed according to a published protocol.22 Briefly, fertilized chicken eggs were cleaned with 70% ethanol and incubated at 37°C under 60% humidity in an egg incubator. On day 3, albumen (2 to 3 mL) was aspirated at the acute pole using a sterile 25-G hypodermic needle to create an air sac directly over the chorioallantoic membrane. A square window (10×10 mm) was then cut in the shell and was sealed with tape. Eggs were returned to the incubator, and, on day 8, the window was opened under sterile conditions and a sterilized gelatin sponge preinfused with VEGF (100 ng/mL) and S66 (20 μmol/L) was placed on the chorioallantoic membrane. On day 11, the vessels surrounding each sponge were counted.
S66 (3-fluoropyridine-4-carboxylic acid (2′,5′-dimethoxybiphenyl-4-yl)amide) was a gift from GSK (GSK1349571A); its chemical identity was confirmed independently by liquid chromatography–mass spectrometry (Leeds) and chemical synthesis. S66 is an abbreviation of “Synta 66” from patent WO 2005/009954.18,19 All other reagents were from Sigma unless specified in the Online Data Supplement.
Averaged data are presented as means±SEM. Data were produced in pairs (test and control) and compared using t tests with statistical significance indicated by an asterisk (P<0.05) and no significant difference by NS (P>0.05). The number of independent experiments is indicated by n. For multiwell assays, the number of replicate wells is indicated by N.
Orai1 Role in Store-Operated and VEGF-Evoked Ca2+ Entry
CRAC channel function was first detected as a component of store-operated Ca2+ entry, which was investigated using an intracellular Ca2+ indicator to detect Ca2+ entry when extracellular Ca2+ was added back to endothelial cells that had been store-depleted using the pharmacological agent thapsigargin (eg, Figure 1a). The Ca2+ add-back signal was suppressed when Orai1 expression was knocked-down by siRNA, as shown by the example data for EPCs (Figure 1a; Online Figures I and II) and the mean data in Figure 1c. An alternative approach for disruption of Orai1 is expression of exogenous R91W mutant of Orai1, which acts as a DN to inhibit endogenous Orai1 and CRAC channel function.13,23,24 DN-Orai1 also inhibited the Ca2+ entry, as shown by comparative analysis in HUVECs (Figure 1b and 1c). The data confirm previous siRNA studies of HUVECs17 and extend the findings to DN-Orai1 and EPCs.
It was hypothesized that the above Ca2+ signals were relevant to the action of VEGF because VEGF caused Ca2+ release from intracellular stores, as shown for example in HUVECs (Online Figure III). In the continuous presence of extracellular Ca2+, VEGF evoked a transient followed by a sustained elevation of the intracellular Ca2+ concentration (Figure 1d), suggesting that there was Ca2+ entry after transient Ca2+ release. Orai1 siRNA reduced both the transient and sustained effects of VEGF by ≈42% (Figure 1d and 1e). Whole-cell patch-clamp recordings revealed that VEGF-evoked ionic current was below the detectable amplitude when using CRAC channel recording conditions in the presence of extracellular Ca2+ (Online Figures IV through VIII), consistent with prior information on endothelial cells.17 The data suggest that Orai1 contributes substantially to VEGF-evoked Ca2+ signals that are mediated by sub-pA ionic currents.
A previous report suggested that TRPC1 also confers VEGF-evoked Ca2+ entry.9 We similarly observed suppression by anti-TRPC1 blocking antibody (Figure 1f) but the effect was relatively small and variable, not achieving statistical significance across all experiments (Figure 1g).
Mechanism of VEGF-Evoked Ca2+ Entry
Previous research suggested that VEGFR2 is the mediator of VEGF-evoked Ca2+ signals.9 Consistent with this mechanism the multikinase inhibitors sorafenib and vatalanib potently suppressed VEGF-evoked Ca2+ rises (Figure 2a; Online Figure IX). Phospholipase Cγ has also been implicated,9 and so a phospholipase C inhibitor U73122 was investigated. U73122 similarly inhibited VEGF-evoked Ca2+ rises (Figure 2a; Online Figure IX). Inositol-1,4,5-trisphosphate generated by phospholipase C was, therefore, presumed to be the messenger evoking Ca2+-release. Studies of other cell types have suggested that the ensuing depletion of Ca2+ stores causes clustering of STIM1 in endoplasmic reticular membranes and that this STIM1 binds to and activates Orai1 channels in the plasma membrane.14 To investigate this mechanism, HUVECs were transfected with eYFP-tagged STIM1 which showed the expected14 microtubule-like localization pattern (Figure 2b). VEGF caused marked clustering of eYFP-STIM1 (Figure 2c). Additional experiments were carried out where endogenous STIM1 was knocked down by RNA interference (Figure 2d; Online Figure I). STIM1 siRNA suppressed VEGF-evoked Ca2+ signals (Figure 2d).
The data suggest that the pathway for VEGF to evoke Orai1-dependent Ca2+ entry is: VEGFR2; phospholipase Cγ; inositol-1,4,5-trisphosphate; inositol-1,4,5-trisphosphate receptor; Ca2+ release from intracellular stores; STIM1 activation and clustering; STIM1 binding to Orai1; Orai1 (CRAC) channel activation (summarized in Online Figure X).
Subcellular Colocalization of VEGFR2 With Orai1
Using specific mouse monoclonal anti-VEGFR2 and rabbit polyclonal anti-Orai1 antibodies (Online Figures II and XI), we investigated the subcellular localizations of endogenous VEGFR2 and Orai1 in HUVECs (Figure 2e). Partial colocalization was detected in the vicinity of the plasma membrane, most obviously when observing in a focal plane above the adherent surface, where the nucleus was located (Figure 2e; Online Figure XI through XIII). The image in Figure 2e was captured from a cell in the presence of VEGF but VEGF did not obviously change the degree of colocalization (Online Figure XII). The data suggest that these proteins share a subcellular compartment, possibly to enhance signaling efficiency.
Orai1 Role in Cell Migration and Tube Formation
A key process regulated by VEGF is endothelial cell migration,1 which can be quantified in modified Boyden chambers in which the cells migrate through 8-μm pores toward VEGF (Figure 3a; Online Figure XIV).1 It was observed that Orai1 siRNA suppressed HUVEC migration, as shown by the example (Figure 3a) and mean data (Figure 3b). A similar but weaker effect of DN-Orai1 was observed (Figure 3b). The data suggest that Orai1 has a positive role in endothelial cell migration and may therefore play a role in angiogenesis.
To investigate the relevance to angiogenesis, we first quantified HUVEC tube and loop formation on Matrigel as an in vitro assay of angiogenic potential.1,25 Examples of the structures formed by the cells are shown in Figure 3c. Disruption of Orai1 by DN-Orai1 inhibited the tube formation (Figure 3c). Therefore, extensive experiments were performed to test the hypothesis that Orai1 has a role in this phenomenon (Figure 3d). Experiments were carried out in test and control pairs where the white bars are mean control data and the black bars to the right are mean test data (Figure 3d). Two different Orai1 siRNAs (Or1.si.1 and Or1.si.2) independently inhibited tube length and loop formation, whereas an inactive siRNA (Or1.neg.) had no effect (Figure 3d). In cells subjected to Orai1 siRNA, normal tube length and formation could be rescued by expression of exogenous wild-type (WT) Orai1 clone (Figure 3d). EPCs were studied less extensively but loop structures formed by the cells were found to be largely prevented by Orai1 siRNA (Figure 3e and 3f).
The data suggest that Orai1 makes a significant contribution to angiogenic behavior of endothelial cells.
Specific Inhibition of Ca2+ Entry by a CRAC Channel Blocker
The above experiments suggest a role of Orai1, but the results may not be explained by a role of CRAC channels because Orai1 could have multiple functions. Therefore, we sought a specific chemical blocker of CRAC channels as a further test of the role of the channels. The patent WO 2005/009954 describes the compound 3-fluoropyridine-4-carboxylic acid (2′,5′-dimethoxybiphenyl-4-yl)amide as an inhibitor of CRAC channels in immune cells. We refer to the compound as S66. It has previously been reported to specifically inhibit CRAC channel-related signals in immune cells with an IC50 of 1.4 to 3.0 μmol/L.18,19
The Ca2+ add-back signal in store-depleted HUVECs was strongly suppressed by S66 (Figure 4a). The effect occurred with high potency, showing an IC50 of 25.5 nmol/L (Figure 4b). Direct comparisons with immune cell Ca2+ entry under the same conditions confirmed that there was higher potency against HUVECs and EPCs (Online Figure XV). Specificity of S66 was indicated by investigation of two other types of Ca2+ channel that are expressed and functional in endothelial cells, TRPC610,11 and TRPV4.26,27 Overexpressed TRPC6 was activated by the lipid 1-oleoyl-2-acetyl-sn-glycerol (Figure 4c) whereas overexpressed TRPV4 was activated by 4α-phorbol-didecanoate (Figure 4d). S66 (5 μmol/L) had no significant effect on these channels (Figure 4c and 4d).
The data suggest that S66 is an inhibitor of endothelial store-operated Ca2+ entry and lacks effect on TRPC6 and TRPV4.
Inhibition of VEGF-Evoked Ca2+ Entry by the CRAC Channel Blocker
S66 was tested against VEGF-evoked Ca2+ signals in the presence of extracellular Ca2+ (Figure 4e and 4f). It caused strong inhibition of sustained Ca2+ entry while lacking effect on the initial transient response (Figure 4e and 4f). These data contrast with the Orai1 siRNA data of Figure 1d and 1e because there was specificity for the sustained Ca2+ response, suggesting that S66 did not affect VEGF receptors or Ca2+-release. S66 also did not affect STIM1 clustering (Online Figure XVI). To investigate the relevance of the action of S66 to other types of endothelial cell, VEGF responses were investigated in EPCs and cultures of saphenous vein endothelial cells (SVECs) from patients undergoing coronary artery bypass graft surgery. As observed in HUVECs, S66 was a strong inhibitor of the sustained VEGF response but not the transient response (Figure 4f). The residual S66-resistant Ca2+ entry in HUVECs might be accounted for by TRPC1 (Figure 1f and 1g). In saphenous vein endothelial cells the S66-resistant component was larger (Figure 4f), as was the TRPC1 contribution (Online Figure XVII).
The data are consistent with CRAC channels mediating a substantial fraction of the sustained Ca2+ entry evoked by VEGF and with the amplitude of this contribution varying across endothelial cell types.
Inhibition of Tube Formation and Angiogenesis by the CRAC Channel Blocker
S66 was also investigated against endothelial cell migration. The cell migration was inhibited by ≈50% with maximum effect at 2 μmol/L S66 (Figure 5a), suggesting that the S66-sensitive mechanism had a positive modulator role. S66 lacked effect on endothelial cell viability (Figure 5b). To investigate the impact on angiogenesis we performed in vitro and in vivo assays. In in vitro assays S66 restricted tube length and reduced the number of loops, conferring 60 to 70% inhibition (Figure 5c and 5d). For in vivo analysis, we used the established angiogenesis assay of the chick chorioallantoic membrane, where new blood vessels form around VEGF-infused sponges.1,22 S66 had striking inhibitory effects on the growth of these vessels (Figure 5e and 5f). The data suggest that S66 was an inhibitor of VEGF-induced endothelial cell migration and angiogenesis without effect on cell viability.
The study suggests that a CRAC channel protein (Orai1) is important for store-operated and VEGF-evoked Ca2+ entry in endothelial cells, with Orai1 disruption leading to reduced endothelial cell migration and tube formation (see the summary diagram of Online Figure X). Because Orai1 may have CRAC channel independent roles we sought to further investigate the hypothesis that CRAC channels are involved by seeking a specific chemical blocker of the channels. S66 was found to be a strong, potent and specific inhibitor of store-operated Ca2+ entry. Importantly, it replicated the effects of Orai1 disruption on sustained endothelial cell Ca2+ entry and migration and tube formation, and inhibited VEGF-evoked angiogenesis in vivo. Suppression of the transient VEGF Ca2+ response by Orai1 siRNA but not S66 may be explained by partial store-depletion resulting from the long-term disruption of CRAC channels or a CRAC channel-independent role of Orai1.
CRAC channels and Orai1 have mostly been associated with immune cell function.13,28 Therefore, inhibitors of CRAC channels and Orai1 are predicted to be immunosuppressive. Our S66 data suggest that it may be possible to minimize such effects because of the higher potency in endothelial cells. Nevertheless, inhibitor effects on immune cell function could be advantageous in conditions such as obesity or cancer where, for example, low-grade long-term inflammation and late-stage aggressive immune responses have deleterious effects.29,30 Although the effect of S66 seems to open new therapeutic potential it is a limitation that we do not yet know its mechanism of action. Our data suggest that it does not affect Ca2+-release (Figure 4e) or STIM1 clustering (Online Figure XVI), putting its site of action downstream and thus perhaps at Orai1 or another CRAC channel component.
Disease in Orai1-deficient patients is severe but apparently limited to immunodeficiency, congenital myopathy and ectodermal dysplasia.16,31 These patients have intact vasculatures and so Orai1 is not obligatory for vasculogenesis in humans. Orai1-deficient mice are either runted and display immune deficiency or die just before or after birth for reasons that are not yet clear.32,33 Such observations from patients and mutant mice are consistent with our data showing suppression but not abolition of endothelial migration and tube formation following Orai1 disruption. That is, we hypothesize that the role of Orai1 in endothelial function is as a positive modulator rather than obligatory factor. It is, therefore, conceivable that there are compensatory mechanisms for Orai1-deficiency, perhaps involving other Orai proteins or other types of Ca2+ channel protein that have roles in VEGF responses and angiogenesis. Indeed, our data are consistent with a contribution of TRPC1 and there is good evidence for roles of TRPC610,11 that may be particularly relevant to slowly developing VEGF effects outside the time frame of our Cai2+ measurements (see Online Figure X). Integration of Orai1 and TRPC1 is indicated to occur, for example, through STIM1 (Online Figure X).34
In summary, positive roles of Orai1 in endothelial cell VEGF Cai2+ responses, migration, and tube formation are indicated. The effects appear to result substantially from the role of Orai1 in CRAC channels because of the additional finding that a specific CRAC channel blocker had mostly similar effects to Orai1 disruption. Unexpected high potency of the blocker at endothelial compared with immune cells suggested, nevertheless, a distinct characteristic of the endothelial CRAC channel and the opportunity for endothelial specificity. In addition to the mechanistic insight provided by this study, there is the suggestion that the process could be an attractive target for therapeutic modulation of angiogenesis in inflammatory disease situations that include cancer and metabolic syndrome associated with obesity.
Sources of Funding
The work was supported by the British Heart Foundation, Wellcome Trust, and Medical Research Council. L.A.W. was supported by a BBSRC-AstraZeneca PhD Studentship, M.S.A. was supported by a scholarship from the Egyptian Ministry of Higher Education, and B.H. was supported by a Scholarship from the University of Leeds and the China Scholarship Council.
In February 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.7 days.
Non-standard Abbreviations and Acronyms
- calcium-release-activated calcium
- R91W dominant-negative Orai1
- endothelial progenitor cell
- human umbilical vein endothelial cell
- Orai1 negative control small interfering RNA
- Orai1 small interfering RNA 1
- Orai1 small interfering RNA 2
- short interfering RNA
- stromal interaction molecule-1
- STIM1 small interfering RNA
- transient receptor potential canonical
- transient receptor potential vanilloid
- vascular endothelial growth factor
- vascular endothelial growth factor receptor-2
- exogenous wild-type Orai1
- Received October 22, 2010.
- Revision received February 21, 2011.
- Revision received February 21, 2011.
- Accepted March 11, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Orai1 is a T-cell membrane protein component of a Ca2+ entry mechanism that is defective in severe combined immune deficiency.
Depletion of intracellular Ca2+ stores is an activation signal for calcium release–activated calcium (CRAC) channels in T cells.
Orai1 is expressed in human umbilical vein endothelial cells, which contain CRAC channel-like signals.
What New Information Does This Article Contribute?
Endothelial Orai1 stimulates Ca2+ entry, cell migration, and tube formation evoked by vascular endothelial growth factor (VEGF).
A specific chemical blocker of CRAC channels inhibits VEGF-evoked Ca2+ entry, endothelial cell migration, and endothelial tube formation in vitro, and angiogenesis in vivo.
Endothelial Ca2+ entry is more sensitive to CRAC channel blockade than immune cell Ca2+ entry.
Although Orai1 and the CRAC channels have been extensively studied in T cells, little is known about the role of Orai1 and CRAC channels in endothelial cell biology. Our studies show that Ca2+ entry evoked by VEGF depends on Orai1. Molecular investigations, including rescue of function by an exogenous Orai1 clone, showed that Orai1 is important in endothelial tube formation driven by VEGF. Reduced capacity for cell migration was implicated as a contributory factor. A substance with previously described specificity but modest potency for modulating immune cell CRAC channels was found to have higher potency at endothelial Ca2+ entry, blocking VEGF-evoked Ca2+ entry but not Ca2+ release. Endothelial cell migration, in vitro tube formation, and VEGF-evoked angiogenesis in vivo were all suppressed, without affecting cell viability. These findings suggest that Orai1 and CRAC channels are relevant to major aspects of endothelial biology: VEGF signaling, endothelial cell migration, and angiogenesis. Targeting Orai1 and CRAC channels with specific and potent chemical modulators may introduce new opportunities for therapeutic approaches.