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(Circulation Research. 2005;96:1282.)
© 2005 American Heart Association, Inc.

Cellular Biology

Angiopoietin-1 Opposes VEGF-Induced Increase in Endothelial Permeability by Inhibiting TRPC1-Dependent Ca² Influx

David Jho, Dolly Mehta, Gias Ahmmed, Xiao-Pei Gao, Chinnaswamy Tiruppathi, Michael Broman, Asrar B. Malik

From the Department of Pharmacology and The Center of Lung and Vascular Biology, The University of Illinois, Chicago.

Correspondence to Dr Asrar B. Malik, Department of Pharmacology, University of Illinois at Chicago, 835 S Wolcott Ave (M/C 868), Chicago, IL 60612. E-mail abmalik{at}uic.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiopoietin-1 (Ang1) exerts a vascular endothelial barrier protective effect by blocking the action of permeability-increasing mediators such as vascular endothelial growth factor (VEGF) through unclear mechanisms. Because VEGF may signal endothelial hyperpermeability through the phospholipase C (PLC)-IP₃ pathway that activates extracellular Ca²⁺ entry via the plasmalemmal store-operated channel transient receptor potential canonical-1 (TRPC1), we addressed the possibility that Ang1 acts by inhibiting this Ca²⁺ entry mechanism in endothelial cells. Studies in endothelial cell monolayers demonstrated that Ang1 inhibited the VEGF-induced Ca²⁺ influx and increase in endothelial permeability in a concentration-dependent manner. Inhibitors of the PLC-IP₃ Ca²⁺ signaling pathway prevented the VEGF-induced Ca²⁺ influx and hyperpermeability similar to the inhibitory effects seen with Ang1. Ang1 had no effect on PLC phosphorylation and IP₃ production, thus its permeability-decreasing effect could not be ascribed to inhibition of PLC activation. However, Ang1 interfered with downstream IP₃-dependent plasmalemmal Ca²⁺ entry without affecting the release of intracellular Ca²⁺ stores. Anti-TRPC1 antibody inhibited the VEGF-induced Ca²⁺ entry and the increased endothelial permeability. TRPC1 overexpression in endothelial cells augmented the VEGF-induced Ca²⁺ entry, and application of Ang1 opposed this effect. In immunoprecipitation studies, Ang1 inhibited the association of IP₃ receptor (IP₃R) and TRPC1, consistent with the coupling hypothesis of Ca²⁺ entry. These results demonstrate that Ang1 blocks the TRPC1-dependent Ca²⁺ influx induced by VEGF by interfering with the interaction of IP₃R with TRPC1, and thereby abrogates the increase in endothelial permeability.

Key Words: endothelial permeability • vascular endothelial growth factor • angiopoietin-1 • Ca²⁺ influx • transient receptor potential channel

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular endothelial growth factor (VEGF) was originally described as vascular permeability factor based on its property of increasing vessel wall permeability.¹ VEGF binds to VEGF receptor-2 (VEGFR2; aka Flk1) to stimulate phospholipase C (PLC)-dependent IP₃ production, thereby increasing the cytosolic Ca²⁺ concentration ([Ca²⁺]_i).² The VEGF-induced rise in [Ca²⁺]_i is the result of release of intracellular Ca²⁺ stores followed by extracellular Ca²⁺ entry. The Ca²⁺ influx portion of the response occurring via activation of plasmalemma Ca²⁺ channels was shown to be critical in signaling the increase in endothelial permeability in response to a variety of permeability-increasing mediators including thrombin.^3,4 VEGF was shown to induce the formation of interendothelial junctional gaps and increase endothelial permeability through a Ca²⁺-dependent pathway.^5–7

Ang1 cooperates with VEGF in the later stages of embryonic angiogenesis to form the mature vascular endothelial barrier.⁸ However, in the adult microvasculature, the binding of Ang1 to the Tie-2 receptor stabilizes endothelial cell interactions with the extracellular matrix and junctional proteins and enhances endothelial barrier function.^8,9 Transgenic mice overexpressing Ang1 in dermal microvessels were resistant to the leakage of the albumin-binding Evans blue dye in response to VEGF and other inflammatory agents.¹⁰ Adenoviral-mediated delivery of Ang1 in adult mouse vascular endothelia markedly reduced the vascular leakage.¹¹ Recombinant Ang1 inhibited both VEGF- and thrombin-induced hyperpermeability in human umbilical vein endothelial cell (HUVEC) monolayers,¹² indicating that Ang1 can directly antagonize the actions of VEGF and thrombin in endothelial cells. These findings raise the possibility that Ang1 has antiinflammatory properties^12,13 in that it prevented the increases in endothelial permeability; however, the signaling pathways responsible for the endothelial barrier protective function have not been elucidated.

The transient receptor potential canonical (TRPC) subfamily of channel-forming proteins, including TRPC1, are important for regulating Ca²⁺ entry in endothelial cells^14,15 and mediating the increase in endothelial permeability.^16–18 Activation of these channels depends on Ca²⁺ store depletion, hence it is termed capacitative Ca²⁺ entry (CCE) or store-operated Ca²⁺ entry.^4,5 Studies showed that TRPC1-mediated Ca²⁺ entry contributed significantly to the thrombin-induced increase in endothelial permeability.^16–18 In addition, endothelial cells expressing TRPC1 demonstrated cytoskeletal changes associated with increased endothelial permeability in response to CCE activation; these cells did not express either TRPC3 or TRPC6.¹⁹

In the present study, we addressed the possibility that Ang1 interferes with the CCE pathway activated by VEGF, thereby mitigating the VEGF-induced increase in endothelial permeability. We observed that Ang1 inhibited the Ca²⁺ entry without affecting the release of intracellular Ca²⁺ stores. Anti-TRPC1 antibody blocked the VEGF-induced CCE and increase in endothelial permeability. TRPC1 overexpression in endothelial cells augmented the VEGF-induced Ca²⁺ entry, and Ang1 opposed this effect. On the basis of the immunoprecipitation data, the endothelial barrier protective effect of Ang1 was attributed to inhibition of the association of IP₃R and TRPC1. These results demonstrate that Ang1 opposes the TRPC1-dependent Ca²⁺ influx induced by VEGF by interfering with the interaction of IP₃R and TRPC1, and thereby prevents VEGF-induced increase in endothelial permeability.

	Materials and Methods

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VEGF₁₆₅ and Ang1 were generously provided by Dr John Rudge, Regeneron Pharmaceuticals (Tarrytown, NY). We used recombinant BowAng1, which contains 4 Ang1 fibrinogen-like domains fused to the human Fc domain which does not interfere with receptor binding or function.²⁰ HBSS, PBS, and trypsin were purchased from GIBCO BRL Life Technologies. FBS was obtained from HyClone, and EGM-2 was purchased from Clonetics. Transwell chamber inserts were obtained from Corning Costar. Fura 2-AM and BAPTA-AM were obtained from Molecular Probes. Protein A/G-agarose beads were obtained from Santa Cruz Biotechnology. ¹²⁵I-BSA tracer was purchased from ICN. Rabbit polyclonal anti-IP3 receptor antibody was obtained from Calbiochem. Rat monoclonal anti-VEGFR2 antibody was purchased from R&D Systems. All other reagents, including rabbit polyclonal TRPC1 antibodies, were purchased from Sigma.

Endothelial Cell Culture and Transfection
Primary HUVECs obtained from Clontech (Palo Alto, Calif) were grown in EGM-2 supplemented with 10% FBS, and passages 3 to 5 were used for all studies. We overexpressed TRPC1 in human dermal microvascular endothelial cells (HMEC-1) obtained from the CDC, because a high transfection efficiency could be achieved in this cell line using described methods.¹⁶

Transendothelial ¹²⁵I-Albumin Permeability
Costar Transwell units were used to determine the permeability of ¹²⁵I-albumin across endothelial cell monolayers.²¹ Transendothelial clearance rate of ¹²⁵I-albumin was calculated from: CL(µL/min)=[activity_AC(cpm/µL) x volume_AC (µL)]/[activity_LC(cpm/µL) x time(min)], in which CL is clearance (volume of luminal chamber fluid cleared of tracer), activity_AC is counts per µL of abluminal chamber sampling, volume_AC is total abluminal chamber volume at the time of sampling, and activity_LC is counts per µL of luminal chamber fluid added at the beginning of the experiment.

Fura-2AM [Ca²⁺]_i Measurement
Free cytosolic Ca²⁺ concentration [Ca²⁺]_i was measured using fura-2AM as described.²¹ For the Ca²⁺ depletion-repletion protocol, endothelial cells were preloaded with fura-2AM in normal Ca²⁺-containing media (1.3 mmol/L) and were placed in Ca²⁺-free media immediately before recording; extracellular Ca²⁺ (1.3 mmol/L) was readded to the media at 300 sec.

IP₃ Production
Cytosolic IP₃ concentrations were determined with the IP₃ Biotrak radioimmunoassay system kit using the standard curve according to instructions from Amersham Pharmacia Biotech. HUVECs grown to confluence on 60-mm culture dishes were washed 3x with PBS and stimulated with 1 µg/mL VEGF for 15 sec, and the reaction was stopped by the addition of ice-cold 15% (v/v) trichloroacetic acid. The scraped endothelial cells were centrifuged for 15 minutes 2000g, and supernatant was washed 3x with 1 mL water-saturated diethyl ether before neutralization to pH 7.5 using NaHCO₃. Total protein was determined using the DC protein assay following the instructions from Bio-Rad Laboratories.

Patch Clamp Experiments
Current recordings were made in the whole-cell configuration at a holding potential of –50 mV using an Axopatch 200B amplifier with pClamp 8.1 software and a Digidata 1322 A/D converter (Axon Instruments).²¹ The extracellular solution contained (in mmol/L): 135 sodium glutamate, 1 MgCl₂, 4 CaCl₂, 10 glucose, and 10 HEPES, pH 7.4 (using NaOH). The pipette solution contained (in mmol/L): 135 sodium glutamate, 10 CsCl, 10 BAPTA, 1 MgCl₂, 1 ATP, 10 HEPES, pH 7.2 (using CsOH). For certain experiments, we used 30 µmol/L 2,5-di(tert-butyl)hydroquinone (BHQ) and 10 mmol/L nonmetabolizable IP3 (F-IP3).

Immunoprecipitation and Western Blotting
Western blotting for PLC was done as described.²² Immunoprecipitation and immunoblotting with anti-IP3 receptor and anti-TRPC1 antibodies were performed as previously described.¹⁸

Statistical Analysis
Two-tailed Student t-test and one-way ANOVA with Bonferroni post-hoc test were used for statistical comparisons. Values are reported as mean±SEM. Differences were considered significant at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang1 Inhibits VEGF-Induced Increases in [Ca²⁺]_i and Endothelial Permeability
VEGF produced an initial [Ca²⁺]_i transient peak followed by a sustained [Ca²⁺]_i plateau below the maximum level (Figure 1A). Coadministration of Ang1 inhibited these VEGF-induced [Ca²⁺]_i changes in a concentration-dependent manner (Figure 1A and 1B). Ang1 also prevented the VEGF-induced increase in transendothelial ¹²⁵I-albumin permeability in a similar concentration-dependent manner (Figure 1C).

View larger version (16K): [in this window] [in a new window]   Figure 1. Ang1 inhibits VEGF-stimulated increases in [Ca2+]i and endothelial permeability in a concentration-dependent manner. A, Ratiometric measurements of [Ca2+]i were made in confluent HUVEC monolayers (Materials and Methods). Various concentrations of Ang1 were coadministered with 1 µg/mL VEGF at the arrow. Experiments made in triplicate are shown as representative tracings; each tracing is the average response of 20 to 40 cells. B, Mean±SEM of steady state ratiometric [Ca2+]i values (n=5). * and ** indicate significant differences compared with controls and to each other (P<0.05). C, Transendothelial (125)I-albumin clearance at 60 minutes was measured in confluent HUVEC monolayers (Materials and Methods), costimulated with 1 µg/mL VEGF and different concentrations of Ang1. Experiments were made 4 times in triplicate (Mean±SEM, n=4). * and ** indicate significant differences compared with controls and to each other (P<0.001).

Ang1 Opposes VEGF-Induced Increase in Endothelial Permeability Independent of PLC Activation
Pretreatment of HUVECs with anti-VEGFR2 antibody, U-73122 (PLC inhibitor), or BAPTA-AM (intracellular Ca²⁺ chelator) prevented the VEGF-induced increases in [Ca²⁺]_i and endothelial permeability (Figure 2A through 2D); the inhibition was similar to that seen with Ang1. However, chelerythrine chloride (pan-PKC inhibitor) failed to prevent either response (Figure 2A through 2D). These results demonstrate the importance of PLC-dependent Ca²⁺ signaling in the mechanism of VEGF-induced increase in endothelial permeability. We also observed that Ang1 failed to prevent VEGF-activated PLC phosphorylation and IP₃ production (Figure 2E and 2F), indicating that Ang1 inhibited the VEGF-mediated increase in endothelial permeability independent of PLC activation.

View larger version (29K): [in this window] [in a new window]   Figure 2. Anti-VEGFR2 antibody, PLC inhibitor U-73122, and BAPTA-AM prevent the VEGF-induced increases in [Ca2+]i and endothelial permeability. A and B, Ratiometric measurements of [Ca2+]i were made (described in Materials and Methods) in confluent HUVEC monolayers stimulated with 1 µg/mL VEGF after 30 minutes pretreatment with anti-Flk1Ab (VEGFR2-neutralizing antibody), U-73122 (PLC inhibitor), BAPTA-AM (intracellular Ca2+ chelator), and cherylethrine (pan-PKC inhibitor). Each representative tracing is the average response of 20 to 40 cells, and experiments were repeated 4 times. C, Mean±SEM of steady state ratiometric [Ca2+]i values (n=5). * indicate differences among groups (P<0.05). D, Endothelial monolayer permeability was assessed by measuring transendothelial 125I-albumin clearance at 60 minutes after treatment in HUVEC monolayers. Solid bars indicate groups treated with 1 µg/mL VEGF after 30 minutes pretreatment with various inhibitors, and open bars indicate groups treated with inhibitor alone. Experiments were repeated 4 times in triplicate. * increase vs baseline value without VEGF (P<0.05). E, Representative Western blot shows tyrosine phosphorylation of PLC-1 in response to VEGF and the effects of Ang1 and U-73122. Experiments were repeated 4 times. F, IP3 production was quantified and normalized to total protein in confluent HUVEC monolayers treated with 1 µg/mL VEGF or 1 µg/mL Ang1 for 15 sec as described in Materials and Methods (Mean±SEM, n=4). * difference among groups (P<0.05).

Ang1-Induced Blockade of Plasmalemmal Ca²⁺ Entry Prevents the Increase in Endothelial Permeability
Because VEGF activates intracellular Ca²⁺ store release and extracellular Ca²⁺ influx under normal Ca²⁺ conditions, we used the Ca²⁺ depletion-repletion protocol to address whether Ang1 inhibits intracellular Ca²⁺ store release or plasmalemmal CCE. Addition of VEGF in nominally Ca²⁺-free medium resulted in an increase in [Ca²⁺]_i attributable to store release alone. The sustained elevation of [Ca²⁺]_i resulting from CCE was absent until the re-addition of extracellular Ca²⁺ (1.3 mmol/L [Ca²⁺]_o). In the absence of VEGF exposure, replenishment of [Ca²⁺]_o had no significant effect on the baseline [Ca²⁺]_i. Interestingly, the magnitude of CCE was decreased when Ang1 was coadministered with VEGF, whereas the initial peak caused by store release was unaffected (Figure 3A). The magnitude of CCE was reduced even when Ang1 was administered at the time of [Ca²⁺]_o re-addition (Figure 3B and 3C). Using the same Ca²⁺ depletion-repletion approach, we observed that Ang1 also inhibited the VEGF-induced endothelial monolayer permeability to ¹²⁵I-albuminregardless of whether Ang1 was coadministered with VEGF or added at the time of [Ca²⁺]_o repletion (Figure 3D).

View larger version (24K): [in this window] [in a new window]   Figure 3. Ang1 inhibits VEGF-induced Ca2+ entry. Ratiometric measurements of [Ca2+]i during extracellular Ca2+ depletion-repletion conditions (see Methods). A, In nominally Ca2+-free media, VEGF (1 µg/mL) induced an initial increase in endothelial [Ca2+]i representing intracellular store release and a secondary rise in [Ca2+]i on readdition of 1.3 mmol/L [Ca2+]o representing plasmalemmal Ca2+ entry. Ang1 (1 µg/mL) reduced Ca2+ entry on readdition of [Ca2+]o without affecting store release. B, Inhibition of VEGF-induced Ca2+ entry was evident when Ang1 was added at the time of [Ca2+]o repletion. Baseline tracing shows [Ca2+]i levels under Ca2+ depletion–repletion conditions in the absence of treatment. Representative tracings depict the average responses of 20 to 40 cells. C, Mean±SEM of steady state ratiometric [Ca2+]i values (n=10). * difference among groups (P<0.05). D, Transendothelial clearance of 125I-albumin at 60 minutes was measured in confluent HUVEC monolayers grown on microporous filters following the same extracellular Ca2+ depletion-repletion protocol. Experiments were repeated 4 times in duplicate. * difference among groups (P<0.001).

Ang1 Dampens VEGF- and Direct IP₃-Activated Inward Ca²⁺ Current
We addressed the effect of Ang1 in inhibiting Ca²⁺ influx by patch clamping in single endothelial cells. We activated the lanthanum-sensitive inward current by either extracellular VEGF administration or direct intracellular application of IP₃ via the patch pipette. Ang1 was added to the medium after the VEGF- or IP₃-induced inward current had reached a steady state. We observed that perfusion of Ang1 in the bath solution resulted in significant reductions in both VEGF- and IP₃-induced inward currents (Figure 4A through 4F).

View larger version (23K): [in this window] [in a new window]   Figure 4. Ang1 inhibits the inward Ca2+ current induced by VEGF and intracellular application of IP3. A and B, After establishing whole cell patch in HUVECs (see Materials and Methods), VEGF (1 µg/mL) was administered to the extracellular milieu. On reaching a steady inward current, lanthanum chloride (La3+, 1 µmol/L) or Ang1 (1 µg/mL) was added to the extracellular solution as indicated by the solid bars. Representative single-cell current tracings are shown. C and D, Direct activation of internal Ca2+ release induced by administration of IP3 to the intracellular milieu resulted in store-operated inward current measured at a holding potential of –50 mV. La3+ or Ang1 was added to the bath solution as indicated by solid bars after the current had reached steady state. Representative current tracings from single cells are shown. E, Mean±SEM depict the steady-state values of current density (n=5). * and ** indicate differences compared with control and to each other (P<0.05). F, Mean±SEM of steady-state values (n=8). * and ** indicate differences compared with control and to each other (P<0.05).

Anti-TRPC1 Antibody Inhibits VEGF-Induced Increases in [Ca²⁺]_i and Endothelial Permeability
Because TRPC1 channel mediates store-operated Ca²⁺ entry in human endothelial cells,¹⁸ we next addressed the involvement of TRPC1 in the mechanism of VEGF-induced increase in endothelial permeability. We used the antibody that binds amino acids 557 to 571 on the S5 pore-forming region of TRPC1 on the extracellular domain and thereby inhibits Ca²⁺ entry via these channels.^17,23 Pretreatment of HUVECs for 30 minutes with anti-TRPC1 antibody significantly reduced the sustained phase of Ca²⁺ entry stimulated by VEGF, such that there was no further inhibition with Ang1 (Figure 5A and 5B). The VEGF-induced increase in endothelial permeability was also inhibited by the anti-TRPC1 antibody, and Ang1 produced no further inhibition (Figure 5C).

View larger version (16K): [in this window] [in a new window]   Figure 5. Anti-TRPC1 antibody prevents the VEGF-induced increases in [Ca2+]i and endothelial permeability. A, Ratiometric [Ca2+]i measurements were made using fura 2-AM and extracellular Ca2+ depletion-repletion conditions in HUVECs (see Materials and Methods). The effects of TRPC1-neutralizing antibodies and Ang1 (1 µg/mL) on the VEGF (1 µg/mL) response are shown. Representative tracings show the average responses of 20 to 40 cells. B, Mean±SEM of steady-state ratiometric [Ca2+]i values (n=10). * and ** indicate differences compared with controls and to each other (P<0.05). C, Transendothelial clearance of 125I-albumin at 60 minutes was measured in HUVEC monolayers grown to confluence on microporous filters. The inhibitory effects of TRPC1-neutralizing antibodies and Ang1 on VEGF-induced permeability are shown. Experiments were repeated 4 times in triplicate. * difference among groups (P<0.001).

Ang1 Counters the Augmentation in VEGF-Induced Ca²⁺ Entry Induced by TRPC1 Overexpression
To address whether Ang1 can interfere with TRPC1 function, we determined the effects of Ang1 in modifying the CCE pathway using endothelial cells (HMECs) in which TRPC1 was overexpressed 3-fold.¹⁶ On VEGF administration to HMECs transfected with the empty vector, we observed the characteristic Ca²⁺ transient in nominally Ca²⁺-free media and the sustained Ca²⁺ entry on replenishment of [Ca²⁺]_o. Coadministration of Ang1 with VEGF in these cells caused a reduction in Ca²⁺ entry (Figure 6A) as described for HUVECs (Figure 3A and 3B). In the TRPC-1-overexpressing HMECs, we observed augmentation of the Ca²⁺ entry with VEGF challenge, and the coadministration of Ang1 blocked this effect (Figure 6B and 6C).

View larger version (20K): [in this window] [in a new window]   Figure 6. Ang1 counters the VEGF-induced Ca2+ entry augmented by TRPC1 overexpression. HMECs were transfected with empty vector or TRPC1 cDNA construct for ratiometric [Ca2+]i measurements using fura 2-AM and extracellular Ca2+ depletion-repletion (see Materials and Methods). VEGF (1 µg/mL) and Ang1 (1 µg/mL) were administered as indicated. A, Each representative tracing is the average response of 15 to 30 cells in HMEC monolayers transfected with pcDNA3.1 vector. B, Representative tracings show the average responses of 15 to 30 cells for TRPC1-overexpressing HMEC. C, Mean±SEM of steady-state ratiometric [Ca2+]i values (n=10). * and ** indicate differences compared with controls and to each other (P<0.05).

Ang1 Inhibits VEGF-Activated Association of IP₃ Receptor with TRPC1
As the interaction between IP₃R and TRPC1 activates store-dependent Ca²⁺ entry,^18,24 we performed immunoprecipitation studies using anti-IP₃R and anti-TRPC1 antibodies to address the possibility that Ang1 interfered with the association of IP₃R with TRPC1. We observed that in HUVEC lysates, VEGF increased the association of IP₃R and TRPC1, and this effect was blocked by the coadministration of Ang1 (Figure 7A and 7B).

View larger version (37K): [in this window] [in a new window]   Figure 7. Ang1 inhibits VEGF-induced association of TRPC1 and IP3R. A and B, Confluent HUVEC monolayers received the indicated treatments for 1 minute before obtaining cell lysates for immunoprecipitation and immunoblotting as described in Methods. Western blots show lysates immunoprecipitated with anti-IP3R, anti-TRPC1, or nonspecific rabbit polyclonal antibodies followed by immunoblotting with anti-IP3R, anti-TRPC1, or anti-IgG heavy chain antibodies as noted. As controls, lysates immunoprecipitated with nonspecific antibodies failed to show TRPC1 or IP3R bands whereas all lysates demonstrated IgG heavy chain bands. Representative blots, repeated 4 times with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We addressed the possible mechanisms by which Ang1 opposes the VEGF-induced increase in endothelial permeability. Based on previous studies suggesting that VEGF increases permeability through a PLC- and Ca²⁺-dependent pathway,^5–7 we have focused on the role of Ang1 in modulating Ca²⁺ entry, and thereby protecting the endothelial barrier. We demonstrated that VEGF increased endothelial permeability through a mechanism involving the PLC-IP₃ pathway and activation of TRPC1-dependent plasmalemmal Ca²⁺ entry. Ang1 inhibited this signaling pathway by opposing the VEGF-induced coupling of TRPC1 and IP₃R without affecting upstream PLC phosphorylation, IP₃ production, or Ca²⁺ release from intracellular stores. Thus, the results demonstrate that Ang1 blocked the TRPC1-dependent Ca²⁺ influx induced by VEGF by interfering with the interaction of TRPC1 with IP₃R, and thereby abrogated the increase in endothelial permeability.

We used various inhibitors to show that the VEGF-induced permeability response required the PLC-dependent Ca²⁺ signaling pathway. Our data showed that the endothelial barrier protective effect of Ang1 could not be ascribed to the inhibition of VEGF-induced PLC phosphorylation or IP₃ production. This led us to investigate the downstream components of the PLC pathway, that is, the IP₃-dependent store release and plasmalemmal Ca²⁺ entry. We observed the complete inhibition of the [Ca²⁺]_i transient by Ang1 when Ca²⁺ release and Ca²⁺ influx were activated in the presence of normal extracellular Ca²⁺ concentration. Under these conditions, the [Ca²⁺]_i transient is more complex because there is simultaneous activation of intracellular Ca²⁺ store release and extracellular Ca²⁺ influx, both of which are coupled to the interaction between IP₃R and TRPC channels.^18,25,26 We also performed whole-cell patch clamp experiments to assess directly the effects of Ang1 on the inward Ca²⁺ current. In these experiments, when VEGF was used to elicit the Ca²⁺ influx and then Ang1 was added, we observed that Ang1 inhibited the lanthanum-sensitive VEGF-induced inward current. These results were consistent with the fura-2AM measurements in which VEGF was first administered and then Ang1 was added, and in which we observed that Ang1 prevented the Ca²⁺ influx.

We made several observations that support the concept that a threshold [Ca²⁺]_i as regulated by the Ca²⁺ entry is an essential requirement for increasing endothelial permeability. We observed that Ang1 modestly reduced the baseline Ca²⁺ influx in endothelial cells bathed in normal Ca²⁺-containing media, but not when bathed in nominally Ca²⁺-free medium. Although in our studies the basal Ca²⁺ entry was low to begin with, such an effect of Ang1 could explain its action in reducing baseline permeability observed in some studies.¹² We also observed that low concentrations of VEGF elicited small Ca²⁺ transients, whereas higher concentrations were needed to trigger the increase in albumin permeability (unpublished observation). That a minimal threshold of Ca²⁺ entry is needed to signal the increase in endothelial permeability was also supported by both the Ang1 concentration-dependent responses and Ca²⁺ depletion-repletion studies. The inhibition of this threshold Ca²⁺ entry by Ang1 was sufficient to prevent the VEGF-induced increase in endothelial permeability.

TRPC channels are considered good candidates for Ca²⁺ entry pathways such as CCE. Based on the importance of TRPC1 in regulating the Ca²⁺ entry-dependent increase in permeability of the human endothelial carrier,^16–18 we addressed the role of TRPC1 in the mechanism of Ang1-induced permeability modulation. We demonstrated through overexpression or inhibition of TRPC1 as well as by studying TRPC1 interaction with IP₃R, that TRPC1 is the likely channel at which Ang1 interferes with the VEGF response. We observed that Ang1 blocked the VEGF-induced interaction of TRPC1 and IP₃R. Our data are consistent with the coupling model of CCE whereby activation of PLC and generation of IP₃ and the resultant TRPC1-IP₃R interaction is required for the activation of Ca²⁺ influx.^24–27 It is possible that PLC can activate CCE independent of its catalytic function, perhaps by facilitating the localization of the essential components of the Ca²⁺ entry pathway at the plasma membrane.²⁸ However, the present results do not support this notion as an explanation of the Ang1 effect because we observed that perfusion of Ang1 in the bath solution resulted in a significant reduction in the IP₃-activated inward current.

Although the present study has focused on Ang1 regulation of TRPC1 activation, we cannot rule out the involvement of other relevant TRPCs. TRPC4 acts as a functional homologue in mouse endothelia to TRPC1 in humans.^14,29 For agonist-induced Ca²⁺ entry in mouse aortic endothelial cells, TRPC4 was essential as either a channel-forming subunit or a constituent required for channel activation.³⁰ Because TRPC1 and TRPC4 can oligomerize,³¹ it is possible that both may be needed for the VEGF-induced Ca²⁺ entry. The importance of TRPC4 in regulation of endothelial permeability in mice is reinforced by our observations that the effects of Ang1 on VEGF-induced Ca²⁺ entry and permeability are mimicked by deletion of the TRPC4 gene in mice (unpublished data). It is also possible that the VEGF-induced activation of Ca²⁺ entry can occur via other members of the TRPC family such as TRPC6 which is activated by PLC-generated DAG.^32,33 However, our data indicate that CCE is required for the VEGF-induced increase in endothelial permeability, and thus it is unlikely that TRPC6, which is not a CCE channel,³⁴ is involved in the VEGF response.

In summary, the present study demonstrates that Ang1 inhibition of TRPC1-dependent Ca²⁺ influx immediately blocked the VEGF-induced increase in endothelial permeability. The endothelial barrier protective effect of Ang1, mediated by preventing the interaction of TRPC1 with IP₃R and the resultant inhibition of CCE, suggests a novel therapeutic approach for inflammatory diseases associated with increased microvascular permeability.

	Acknowledgments

 
This work was supported by National Institutes of Health grants T32 HL07829, HL45638, HL60678, and HL71794. Portions of this study were previously published in abstract form. We also thank Dr John Rudge for his input during these studies.

	Footnotes

 
Original received July 3, 2003; first resubmission received February 17, 2004; second resubmission received April 26, 2004; third resubmission received March 21, 2005; revised third resubmission received May 17, 2005; accepted May 18, 2005.

	References

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