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Circulation Research. 2003;93:438-447
Published online before print July 24, 2003, doi: 10.1161/01.RES.0000088358.99466.04
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(Circulation Research. 2003;93:438.)
© 2003 American Heart Association, Inc.

Cellular Biology

Angiotensin II Type 2 Receptor Inhibits Vascular Endothelial Growth Factor–Induced Migration and In Vitro Tube Formation of Human Endothelial Cells

Ralf Benndorf, Rainer H. Böger, Süleyman Ergün, Anna Steenpass, Thomas Wieland

From the Institut für Experimentelle und Klinische Pharmakologie (R.B., R.H.B., A.S.), Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany; Institut für Anatomie (S.E.), Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany; and Institut für Pharmakologie und Toxikologie (T.W.), Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany.

Correspondence to Dr Ralf Benndorf, Institut für Experimentelle und Klinische, Pharmakologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. E-mail benndorf{at}uke.uni-hamburg.de


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Endothelial cell migration and tube formation in response to vascular endothelial growth factor (VEGF) play an important role in the process of angiogenesis. Recent data indicate that angiotensin type 2 (AT2) receptor stimulation is antiangiogenic. Therefore, we studied the effect of angiotensin II (Ang II) on VEGF-induced migration and in vitro tube formation of human endothelial cells. Ang II inhibited VEGF-induced migration of EA.hy926 cells, human coronary artery (HCA) and human dermal microvascular (HDM) endothelial cells (ECs) as well as tube formation by HDMECs. The AT2 receptor antagonist PD123,319 but not the AT1 receptor antagonist losartan blocked the inhibitory effect of Ang II. The inhibitory effect of Ang II on VEGF-induced migration of endothelial cells was mimicked by the specific AT2 receptor agonist CGP-42112A. The phosphorylation of Akt and its downstream effector endothelial NO synthase (eNOS) is pivotal to VEGF-induced angiogenesis. We therefore investigated the effect of Ang II on VEGF-induced Akt and eNOS phosphorylation. Ang II diminished the VEGF-induced phosphorylation of Akt and eNOS in endothelial cells, whereas the autophosphorylation of VEGF receptors was unaffected. CGP-42112A again mimicked and PD123,319 but not losartan blocked the inhibitory effect of Ang II. Treatment of endothelial cells with pertussis toxin (PTX) totally abolished the AT2 receptor–mediated inhibition of VEGF-induced endothelial cell migration and blocked the inhibition of Akt and eNOS phosphorylation. In conclusion, this study indicates that AT2 receptor stimulation inhibits VEGF-induced endothelial cell migration and tube formation via activation of a PTX-sensitive G protein. These findings may explain the reported antiangiogenic properties of the AT2 receptor.


Key Words: angiogenesis • migration • angiotensin II • AT2 receptor


*    Introduction
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 up arrowAbstract
 *Introduction
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Angiogenesis is a characteristic feature of embryonic development and wound healing, but is also involved in pathologies such as ischemic heart disease, cancer, or chronic inflammation. This tightly regulated process includes movement of endothelial cells out of existing vessels, migration toward angiogenic stimuli, proliferation, and formation of new endothelial tubes.1 Vascular endothelial growth factor (VEGF) promotes in vitro migration and tube formation of endothelial cells2,3 and plays a key role in hypoxia-induced angiogenesis.4 It has been established that VEGF-induced angiogenesis is NO-dependent. For example, endothelial NO synthase (eNOS) inhibitors block VEGF-induced endothelial cell migration, proliferation, and tube formation in vitro as well as VEGF-induced angiogenesis in vivo.3,5 VEGF is known to stimulate Akt-dependent phosphorylation of eNOS (Ser-1177) Ca2+-independently via its receptor Flk-1/KDR.6 This results in an activation of eNOS and an increased NO production of endothelial cells. Hence, VEGF-induced phosphorylation of Akt (Ser-473) and eNOS (Ser-1177) plays a key role in VEGF-stimulated angiogenesis. Further factors have been implicated in the regulation of angiogenesis. Among these, angiotensin II (Ang II), the main effector peptide of the renin-angiotensin system, is of particular interest. Ang II can stimulate angiogenesis in vivo and endothelial cell proliferation in vitro.7–9 The biological effects of Ang II are mediated by angiotensin II receptors, of which two major mammalian subtypes, AT1 receptor and AT2 receptor, have been identified.10–12 The AT1 receptor is abundantly expressed in cardiovascular tissue13 and most of the cardiovascular actions of Ang II are attributed to this subtype. AT1 receptor induces migration of smooth muscle cells (SMCs) and monocytes and promotes endothelial cell proliferation.9,14,15 In vivo, the AT1 receptor was shown to promote hypoxia-induced angiogenesis and microvascular angiogenesis,7,8 which may be due to an AT1 receptor–mediated upregulation of VEGF expression.16 In contrast to the AT1 receptor, the AT2 receptor is expressed at low density in the adult,13 but is upregulated in cardiovascular tissue in response to ischemia and inflammation.17 It has been frequently reported that the AT2 receptor exerts AT1 receptor antagonistic actions, eg, inhibition of endothelial cell proliferation or inhibition of AT1 receptor–induced SMC migration in AT2 receptor transfected SMCs.9,18 Furthermore, the AT2 receptor may act as a direct AT1 receptor antagonist.19 Most recently, an antiangiogenic effect of the AT2 receptor in hypoxia-induced angiogenesis has been reported.20 However, the effect of Ang II on endothelial cell migration and in vitro tube formation has not been explored in detail so far. In an in vitro assay, Ang II did not affect basal migration of bovine aortic endothelial cells but abolished lisinopril-induced migration.14 We therefore hypothesized that the reported AT2 receptor–mediated antiangiogenic effect may be due to VEGF-antagonistic actions on endothelial cell migration and tube formation. The data presented herein, provide evidence that AT2 receptor–mediated activation of a pertussis toxin (PTX)–sensitive G protein inhibits VEGF-induced endothelial cell migration and tube formation.


*    Materials and Methods
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 *Materials and Methods
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Materials
Chemicals and reagents were obtained from Sigma Chemical Co, unless noted otherwise. The AT1 receptor antagonist losartan was generously donated from MSD Sharp & Dome (Haar, Germany). Recombinant vascular endothelial growth factor was purchased from PeproTech Inc, and PTX was from List Biological Laboratories Inc. Anti–phospho-eNOS antibody (Ser-1177), anti–phospho-Akt (Ser-473), and anti-Akt antibody were from Cell Signaling Technology. Anti-eNOS antibody was from Transduction Laboratories. Anti-phosphotyrosine (clone 4G10) antibody was from Upstate. Anti–Flk-1/KDR antibody was from Santa Cruz Biotechnology, and anti–ß-tubulin was from Sigma.

Cell Cultures
The experiments were performed using human coronary artery (HCA) and human dermal microvascular (HDM) endothelial cells (ECs) and the permanent human endothelial cell line EA.hy926.21 HDMECs and HCAECs were purchased from Promocell, whereas EA.hy926 cells were a kind gift from Dr Edgell (University of North Carolina, Chapel Hill, NC). HCAECs and HDMECs were cultured in culture flasks (25 cm2) in ready-for-use EC medium (Promocell) containing 5% FCS, 10 ng/mL recombinant epidermal growth factor, 1 µg/mL hydrocortisone, 500 µg/mL gentamicin, and 500 ng/mL amphotericin B at 37°C in humidified air with 5% CO2. Cells in passages 2 to 4 (HDMECs) or 3 to 6 (HCAECs) were used for all assays. EA.hy926 cells were cultured as previously described21 in Eagle medium with 10% FCS and 5 mmol/L hypoxanthine, 0.8 mmol/L thymidine, and 20 mmol/L aminopterine.

Cell Migration Assay
Transwell cell migration assays were performed as previously described,22 using a 96-well Boyden chemotaxis apparatus (Neuroprobe). Briefly, polycarbonate filters with 8 µm (Neuroprobe) were coated with 100 µg/mL type I collagen (Cohesion), diluted in 20 mmol/L acetic acid overnight, washed with phosphate buffered saline, and placed on lower chambers of the chemotaxis apparatus that contained samples. Samples (as specified under Results) were solved in basal endothelial medium (Promocell; HCAECs and HDMECs) or Eagle medium (EA.hy926) with 0.1% bovine serum albumin (BSA). Cells were lifted with 0.05% trypsin/ 0.53 mmol/L EDTA, washed, and 2x104 cells suspended in 50 µL basal endothelial medium (HCAECs and HDMECs) or Eagle Medium (EA.hy926) with 0.1% BSA were pipetted into each upper chamber. The filled apparatus was incubated for 4 (EA.hy926) or 5 hours (HCAECs and HDMECs) at 37°C in humidified air with 5% CO2. After incubation the filter was removed from the apparatus, and cells were fixed with methanol and stained with a Giemsa solution (Dade Behring). Nonmigrated cells were removed from the upper surface of the insert with a cotton swab. The number of migrated cells was counted in six randomly chosen fields under x400 magnification and averaged. All experiments were performed in triplicate or quadruplicate and each experiment was repeated at least two times. Migration was expressed as a percentage of basal cell migration. In some experiments cells were preincubated with PTX (0.1 to 100 ng/mL) for 24 hours before being subjected to migration assays.

Endothelial Tube Formation Assay
To study the effects of Ang II on morphogenetic events of endothelial cells during capillary like tube formation in vitro, 3-dimensional type I collagen gels (Vitrogen 100) were prepared in 48-well cluster tissue culture dishes (Costar) as described previously.23 HDMECs were seeded onto solidified gels at a concentration of 2x104/well in 300 µL of MV medium containing 5% FCS. After the cells reached confluence, the medium was replaced by basal medium containing 5% FCS without further supplements. Twenty-four hours later, factors including VEGF, Ang II, AT1 receptor antagonist losartan, and AT2 receptor antagonist PD123,319 alone or in combination were added to the cells for 3 days. Photographs were taken by phase contrast microscopy (Zeiss) at day 3 of treatment.

Western Blot Analysis of Ang II Receptor Protein Expression in Endothelial Cells
HCAECs, HDMECs, and EAhy.926 cells were lysed in Triton/Nonidet P-40 lysis buffer (0.5% Triton X100, 0.5% Nonidet P-40, 10 mmol/L Tris, pH 7.5, 2.5 mmol/L KCl, 150 mmol/L NaCl, 30 mmol/L ß-glycerophosphate, 50 mmol/L NaF, 1 mmol/L Na3Vo4, and 0.1% protease inhibitor mix), scraped of the dish, and centrifuged at 10 000g for 10 minutes. The supernatant was used as a total cell lysate and analyzed for protein concentration by the Bradford method (BioRad). Equal amounts of cellular proteins (50 µg/lane) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Biosciences). The blots were blocked with 3% skimmed milk in Tris-buffered saline/Tween 20 (TBST) and incubated with rabbit anti-human AT1 (N-10, Santa Cruz Biotechnology) and goat anti-human AT2 (C-18, Santa Cruz) antiserum at a dilution of 1:300 for 12 hours at 4°C. The blots were subsequently washed 3x for 10 minutes with TBST. Bound antibodies were detected by peroxidase-conjugated anti-rabbit (dilution 1:4000) and anti-goat IgG (1:3000) antibodies and the ECL system (Amersham Bioscience). Specificity of bands was assured by incubation of the antisera with AT1-(N-10 P)/AT2-(C-18 P) receptor–specific blocking peptides.

Western Blot Analysis of Akt (Ser-473) and eNOS (Ser-1177) Phosphorylation and Tyrosine Phosphorylation of the Flk-1 Receptor in Endothelial Cells
Western blot analysis of Akt and eNOS phosphorylation and tyrosine phosphorylation of the Flk-1 receptor was performed as described previously.24 In brief, confluent HCAECs and EAhy.926 cells were serum-starved for 18 hours either in basal endothelial medium containing 0.1% BSA or in basal endothelial medium with 0.1% BSA and 1 ng/mL PTX. Cells were then stimulated with VEGF and Ang II or CGP-42112A for 20 minutes in presence or absence of Ang II receptor antagonists as specified under results. Cells were lysed as described above. Equal amounts of cellular proteins (50 µg/lane) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. After incubation in blocking solution (5% bovine serum albumin, phosphate-buffered saline, pH 7.5, 0.1% Tween 20), membranes were incubated with appropriate primary antibodies: anti–phospho-Akt (Ser-473; dilution 1:1000), anti–phospho-eNOS (Ser-1177; dilution 1:1000), anti-phosphotyrosine (clone 4G10; dilution 1:500) for 2 hours at room temperature. Bound primary antibodies were visualized as described above. After stripping, the membranes were reprobed with anti-Akt (dilution 1:1000), anti-eNOS (dilution 1:2000), or anti–Flk-1/KDR (dilution 1:200) antibodies to uncover loading differences. To examine the influence of Ang II stimulation on Flk-1/KDR expression, endothelial cells were incubated with 10-8 mol/L Ang II for up to 6 hours and processed as described above. VEGF receptors were visualized by an anti-Flk-1/KDR (dilution 1:200) antibody. To adjust loading differences, membranes were reprobed with an anti–ß-tubulin (dilution 1:750) antibody. Densitometric quantification of immunoblots was performed using ZeroDScan software. Results were normalized by arbitrarily setting the densitometric value obtained in control cells to 100%.

Detection of Apoptotic Cells
EA.hy926 cells, HCAECs, and HDMECs were seeded on collagen-coated 8-well cover slides (Laboratory Tek) and cultured until subconfluent. Cells were then treated as described for the cell migration and tube formation protocol. After 24 and 48 hours, cells were washed with phosphate-buffered saline and nuclear morphology was assessed by staining of the genomic DNA using with Hoechst 33258 for 10 minutes. Cells were examined and photographs were taken using a fluorescence microscope (Zeiss). Apoptotic cells were distinguished by their characteristic patterns of nuclear condensation and cytoplasmic rounding. The number of apoptotic and nonapoptotic cells was counted in four randomly chosen fields/well (at least 600 cells/well) under x200 magnification. The number of apoptotic cells is expressed as percentage of total cell count and related to basal cell apoptosis (percentage of control). Results are given as mean±SD of two separate experiments performed in triplicate.

Statistical Analysis
All data are expressed as mean±SD. All comparisons were carried out by ANOVA. Post-hoc analysis (Bonferroni) allowed identification of significant differences between groups. Probability values were considered significant at P<0.05.


*    Results
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 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
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Detection of Ang II Receptors in Endothelial Cells
Western blots using AT1- and AT2 receptor–specific antibodies were performed (Figure 1). AT1 receptor and AT2 receptor were detected as a 41-kDa (Figure 1A) and a 60-kDa (Figure 1B) protein, respectively. All cell types (HCAECs, HDMECs, and EA.hy926 cells) expressed significant amounts of AT1 receptor and AT2 receptor protein. The AT1 receptor however is apparently more abundant in EA.hy926 cells than in primary human endothelial cells.



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  		Figure 1. Expression of angiotensin II receptors in endothelial cells. AT1 (A) and AT2 (B) receptor proteins were detected in HDMECs (lanes 1 and 4), HCAECs (lanes 2 and 5), and EA.hy926 cell (lanes 3 and 6) lysate (50 µg of protein) by polyclonal antibodies in the absence (lanes 1, 2, and 3) or presence (lanes 4, 5, and 6) of specific blocking peptides. Lane 3 in panel A shows a second specific band that most likely results from proteolytic cleavage of the AT1 receptor.

Influence of Ang II on Basal Migration of Endothelial Cells
Ang II (in presence or absence of Ang II receptor antagonists) did not modify basal transwell migration of primary endothelial cells (HCAECs/HDMECs; data not shown). In contrast, high concentrations of Ang II (10-5 mol/L) significantly induced basal migration of EA.hy926 cells (as percentage of control 122.9±8.2%; P<0.05 versus control). This effect was apparently mediated through AT1 receptor stimulation as it could be blocked by losartan (101.5±5.7%; P<0.05 versus Ang II alone). In the presence of PD123,319 (10-7 mol/L) the minimum concentration of Ang II to induce motogenesis was significantly reduced to 10-8 mol/L (121.5±6.3%; P<0.05 versus control). PD123,319 did not alter the maximum motogenic effect induced by Ang II. The motogenic effect of 10-7 mol/L Ang II was not seen in the presence of both antagonists (10-7 mol/L each). When losartan (10-7 mol/L) was applied alone, Ang II (10-7 mol/L) exerted an inhibitory effect on EA.hy926 cell migration (83.9±6.5%; P<0.05 versus control). This effect was not seen in the additional presence of PD123,319 (10-7 mol/L). The AT1 receptor– and AT2 receptor–specific antagonists (10-7 mol/L each) losartan and PD123,319, applied alone, did not alter basal EA.hy926 cell migration.

Influence of Ang II on VEGF-Induced Endothelial Cell Migration
Ang II significantly inhibited the motogenic effect of VEGF (50 ng/mL) in all studied cell types in a concentration-dependent manner. A significant inhibition was observed at 10-8 and 10-9 mol/L Ang II in EA.hy926 cells (Figure 2A; P<0.05) and primary endothelial cells (Figure 2B; P<0.01), respectively. The inhibitory effect was maximal at 10-7 mol/L and 10-8 mol/L Ang II for EA.hy926 and primary endothelial cells, respectively.



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  		Figure 2. Angiotensin II (Ang II) inhibits the VEGF-induced migration of EA.hy926 cells (A) and primary endothelial cells (B) in a concentration-dependent manner. Transwell migration of EA.hy926 cells and HCAECs was stimulated with VEGF (50 ng/mL) in the absence or presence of increasing concentrations (10-11 to 10-6 mol/L) of Ang II. Results are expressed as mean±SD of 2 separate experiments performed in quadruplicate. §P<0.05, #P<0.01, *P<0.001 vs Control.

The AT2 receptor agonist CGP-42112A (10-8 mol/L) mimicked the effect of Ang II on VEGF-induced endothelial cell migration (Figure 3). The AT1 receptor antagonist losartan (10-7 mol/L) did not affect the inhibitory action of Ang II on VEGF-induced transwell migration. In contrast, PD123,319 (10-7 mol/L) significantly diminished the inhibitory effect of Ang II in all cell types. Combination with losartan did not alter the effect of PD123,319 alone.



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  		Figure 3. Angiotensin II (Ang II) and the specific AT2-receptor agonist CGP-42112A diminish VEGF-induced migration of EA.hy926 cells and primary endothelial cells. Transwell migration of EA.hy926 cells (A), HCAECs (B), and HDMECs (C) was measured in the absence and presence of VEGF (50 ng/mL). Additionally, Ang II (10-8 mol/L [B and C] or 10-7 mol/L [A]), CGP-42112A (10-8 mol/L), the AT1 receptor antagonist losartan (Lo, 10-7 mol/L), or the AT2 receptor antagonist PD123,319 (PD, 10-7 mol/L) was present if indicated. Results are expressed as mean±SD of 3 separate experiments performed in triplicate. #P<0.001 vs Control; *P<0.01, vs VEGF alone; §P<0.05 vs VEGF+Ang II; and &P<0.05 vs VEGF+CGP.

Influence of Ang II on VEGF-Induced Tube Formation
To test whether the influence of Ang II can be also observed in an assay much closer to the in vivo situation of angiogenesis than transwell migration in vitro tube formation of HDMECs was studied. HDMECs were cultured on collagen gel until they reached subconfluence. Endothelial tube formation was induced by treatment with VEGF (50 ng/mL) (Figure 6A). No tube formation was observed when the cells were exposed to medium only (Figure 6B). The addition of Ang II (10-8 mol/L) did not induce endothelial tube formation (Figure 6C). However, VEGF-induced formation of endothelial tubes was blocked when VEGF and Ang II were applied simultaneously (Figure 6D). This effect of Ang II was absent in the presence of the AT2 receptor–selective antagonist PD123,319 (10-7 mol/L; Figure 6E). In contrast, the simultaneous application of VEGF, Ang II plus losartan (10-7 mol/L) did not alter the inhibitory influence of Ang II (Figure 6F) on VEGF-induced tube formation. Interestingly, slightly enhanced tube formation was detectable when VEGF and PD123,319 were applied simultaneously (Figure 6G), whereas the application of PD123,319 alone did not have any morphogenic effects on endothelial cells (Figure 6H).



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  		Figure 6. Endothelial tube assay on 3-D collagen gel using HDMECs. A, Induction of capillary-like endothelial tubes by VEGF (50 ng/mL). B, Cells treated with basal medium without supplement only. C, Treatment with angiotensin II (Ang II; 10-8 mol/L) alone did not induce tube formation. D, Combined application of VEGF plus Ang II (10-8 mol/L) blunted the stimulatory effect of VEGF. E, Ang II–induced inhibition of endothelial tube formation was blocked by treatment of HDMECs with VEGF plus Ang II and the AT2 receptor antagonist PD123,319 (10-7 mol/L). F, Inhibition of tube formation by Ang II was not reversed by application of VEGF plus Ang II and the AT1 receptor antagonist losartan (10-7 mol/L). G, A weakly enhanced tube formation and increased density of endothelial tubes were observed by treatment of endothelial cells with VEGF plus PD123,319. H, No effects on endothelial morphogenesis were seen by application of PD123,319 alone.

Effect of PTX on Migration of Endothelial Cells and the AT2 Receptor–Mediated Inhibition of VEGF-Stimulated Migration
It has been previously reported that coupling to Gi/o proteins may be of major importance for AT2 receptor signaling.25 To examine the possible involvement of Gi/o proteins in AT2 receptor–mediated inhibition of VEGF-induced and basal endothelial cell migration, we treated EA.hy926 cells, HCAECs, and HDMECs with increasing concentrations of PTX (0.1 to 100 ng/mL) for 24 hours. Cells were subsequently subjected to migration assays and migration was expressed as percentage of control. Surprisingly, PTX treatment was associated with a reduction in basal migration of all cell types (Figure 4). As shown for EA.hy926 cells, the response to PTX was concentration-dependent and reached statistical significance (P<0.01; Figure 5) at a concentration of 1 ng/mL PTX. The inhibitory effect was maximal at a concentration of 10 ng/mL PTX (HCAECs, 39.3%; HDMECs, 37.2%; EA.hy926 cells, 21.5% of control). In accordance with previously published data26,27 even high doses of PTX did not counteract the motogenic effect of VEGF on endothelial cells (Figures 4 and 5Down). Nevertheless, treatment of endothelial cells with PTX completely abolished the Ang II–induced inhibition of VEGF-stimulated endothelial cell migration (Figure 4). Interestingly, treatment of EA.hy926 cells with PTX (1 ng/mL) mimicked the effect of PD123,319 on Ang II–induced migration of untreated EA.hy926 cells and sensitized these cells to the pro-motogenic effect of Ang II (data not shown). In PTX-treated EA.hy926 cells, concentrations of 10-7 mol/L Ang II and higher significantly induced migration (P<0.05).



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  		Figure 4. Treatment of endothelial cells with PTX abrogates the AT2 receptor–mediated inhibition of VEGF-induced transwell migration. EA.hy926 cells (A), HCAECs (B), and HDMECs (C) were either treated with PTX (1 ng/mL for 24 hours) or remained untreated. Cells were subsequently subjected to transwell migration assays. Control denotes basal transwell migration of untreated endothelial cells. Concentration of angiotensin II (Ang II) ranged from 10-8 (B and C) to 10-7 mol/L (A). Results are expressed as mean±SD of 3 separate experiments performed in triplicate. #P<0.001 vs Control; *P<0.01 vs VEGF alone; §P<0.01 vs Control; and $P<0.05, and +P<0.01 vs PTX alone.



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  		Figure 5. PTX inhibits basal migration of EA.hy926 cells in a concentration-dependent manner without affecting the stimulatory effect of VEGF. EA.hy926 cells were treated with increasing concentrations of PTX (0.1 to 100 ng/mL) for 24 hours or remained untreated ({infty}). Subsequently, cells were subjected to transwell migration assays. Basal (filled squares) and VEGF (50 ng/mL; filled triangles)-induced migration are shown. Results are expressed as mean±SD of 3 separate experiments performed in triplicate. §P<0.01, *P<0.001 PTX vs Control; and $P<0.01, #P<0.001 PTX+VEGF vs VEGF.

Effect of Ang II and CGP-42112A on VEGF Signaling and Flk-1/KDR Expression in Endothelial Cells
Recent data indicate that AT2 receptor stimulation may inhibit phosphorylation of Akt at Ser-473,28 thereby providing a hypothetical opportunity by which the AT2 receptor could interfere with VEGF signaling in endothelial cells. To elucidate underlying mechanisms responsible for the antiangiogenic effect of the AT2 receptor, we examined the effect of Ang II and CGP-42112A on VEGF-induced Flk-1/KDR autophosphorylation as well as Akt (Ser-473) and eNOS (Ser-1177) phosphorylation in EA.hy926 cells and HCAECs. In both cell types, Ang II or CGP-42112A had no effect on Flk-1/KDR receptor autophosphorylation and Ang II stimulation for up to 6 hours did not change the expression of Flk-1/KDR receptors in these endothelial cells (data not shown). Nevertheless, Ang II (10-8 mol/L) as well as CGP-42112A (10-8 mol/L) significantly diminished the VEGF-induced Akt and eNOS phosphorylation (Figure 7). Moreover, PD123,319 (10-7 mol/L) and PTX treatment abolished the observed inhibitory effects of Ang II and CGP-42112A. In contrast, losartan (10-7 mol/L) exhibited no influence on the inhibitory effect of Ang II.



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  		Figure 7. Angiotensin II (Ang II) and CGP-42112A (CGP) diminish VEGF-induced phosphorylation of Akt and eNOS. HCAECs were stimulated for 20 minutes with VEGF (50 ng/mL) in the presence or absence of Ang II (10-8 mol/L) or the specific AT2 receptor agonist CGP-42112A (10-8 mol/L). Ang II receptor subtype specificity was investigated using the Ang II receptor antagonist losartan (10-7 mol/L) and PD123,319 (PD; 10-7 mol/L). If indicated, cells were treated with pertussis toxin (PTX; 1 ng/mL, 18 hours) before the experiments. Phosphorylated Akt and eNOS were visualized by immunoblotting with antibodies specific for the phosphorylated protein (p-Akt; p-eNOS). Detection of the unphosphorylated protein served as loading control (Akt; eNOS). A representative experiment is shown in panel A. Results were normalized by arbitrarily setting the densitometric value obtained from unstimulated cells to 100%. Mean±SD of n=4 independent experiments is shown (B). *P<0.05 vs control; +P<0.05 vs VEGF.

Effect of Ang II and PTX on Endothelial Cell Apoptosis
Under certain conditions, Ang II can induce apoptosis in human endothelial cells.29,30 In addition, the concentration-dependent inhibition of basal endothelial cell migration by PTX may result from the induction of apoptosis. We therefore tested whether experimental conditions applied for the transwell migration assay induce apoptosis in HCAECs, HDMECs, and EA.hy926 cells. Cells were stimulated with VEGF (50 ng/mL) in the absence or presence of Ang II (10-8 mol/L) or with Ang II alone for 5 hours plus 19 hours with medium or with 100 ng/mL PTX for 24 hours. Thereafter, nuclear fragmentation was visualized by Hoechst 33258 staining. No alterations in endothelial cell apoptosis were induced by Ang II or PTX as compared with VEGF-treated or untreated control cells (data not shown). As the stimulation of HDMECs with VEGF and/or Ang II was considerably longer during the tube assay, these cells were additionally incubated with VEGF in the presence or absence of Ang II (10-8 mol/L) for 48 hours or remained untreated (Figure 8). As shown in Figure 8A, 6.81±1.92% of untreated HDMECs exhibited an apoptotic phenotype. VEGF (Figure 8B) significantly reduced apoptosis of HDMECs (1.95±0.81%; P<0.01 versus control, n=6). When VEGF and Ang II (10-8 mol/L) were applied simultaneously, no alteration of the amount of apoptotic cells compared with VEGF-treated HDMECs was observed (Figure 8C; 2.15±0.83; P<0.01 versus control, n=6).



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  		Figure 8. Detection of apoptosis of HDMECs by Hoechst 33258 staining. HDMECs remained untreated (A) or were treated with VEGF (B; 50 ng/mL) or VEGF plus angiotensin II (Ang II) (C, 10-8 mol/L) for 48 hours, as described in the Materials and Methods section. Representative micrographs of Hoechst 33258–stained cells are shown. Arrows denote nuclear condensation of apoptotic cells.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The role of Ang II in angiogenesis, especially ischemia-induced angiogenesis, has been extensively studied applying different in vivo models, and there is consensus that Ang II promotes angiogenesis via the AT1 receptor.7,8,31,32 Hypoxia-induced angiogenesis mainly involves an increase in VEGF and eNOS protein content.1 New lines of evidence further indicate that the angiogenic properties of Ang II may be due to an AT1 receptor–mediated increase in VEGF protein expression and an upregulation of VEGF receptor (KDR) expression.16,33,34 In line with these data, it has been previously reported that pretreatment with Ang II potentiates the VEGF-induced in vitro tube formation of bovine retinal endothelial cells (BRECs).34 A most recent publication however, reported an antiangiogenic effect of Ang II mediated by the AT2 receptor in ischemia-induced angiogenesis.20 Furthermore, AT2 receptor–mediated antiangiogenic actions in microvascular growth have been described.7 In this study, we therefore investigated the effect of Ang II on VEGF-induced and basal endothelial cell migration and in vitro tube formation. Our results clearly demonstrate that AT2 receptor stimulation counteracts the VEGF-induced endothelial cell migration and in vitro tube formation pathway in all three cell types under study. The apparent discrepancy of our findings to those of reported from BRECs is most likely due to the different experimental design.34 Otani and coworkers treated BRECs with Ang II for 24 hours. Thereafter, cells were subjected to in vitro tube formation assays in the presence of VEGF but in absence of Ang II. Thus, the potentiated response to VEGF of the Ang II treated cells is most likely caused by the observed increase in KDR expression, which is attributed to AT1 receptor stimulation. In contrast, we performed tube formation assays using untreated HDMECs to which medium containing VEGF and Ang II±Ang II receptor antagonists was added simultaneously. This experimental design disclosed the antiangiogenic properties of Ang II. Our data demonstrate that the antimotogenic effects of Ang II in all three cell types are exclusively mediated through an AT2 receptor–mediated signaling. It was mimicked by the specific AT2 receptor agonist CGP-42112A and was affected by the AT2 receptor–selective antagonist PD123,319 but not by the AT1 receptor–specific losartan. As migration and tube formation of endothelial cells are crucial steps in the angiogenic process,1 the herein observed inhibition of the VEGF-induced cell migration may at least in part account for the antiangiogenic properties of the AT2 receptor.20 Our findings may be of particular importance in ischemia-associated angiogenesis, as vascular AT2 receptor expression increases in response to ischemia or vascular injury.17 The efficient migration of endothelial cells toward chemotactic stimuli may be critical for the resolution of denudation injuries to the vessel wall that regularly occur in connection with PTCA or bypass surgery.35 There are data from other cell types that support a general antimotogenic action of AT2 receptors. In vascular smooth muscle cells (SMCs), which do not express AT2 receptors,14,15,18 Ang II induces SMC migration through AT1 receptor stimulation. However, when recombinant AT2 receptor was expressed in SMCs, antimotogenic and AT1 receptor–antagonistic effects of the AT2 receptor were observed.18

Meanwhile, a variety of publications have established that VEGF-induced angiogenesis is NO-dependent. For example, VEGF-induced endothelial cell migration and tube formation in vitro can be blocked by eNOS inhibitors.3,36 Moreover, it has been demonstrated that VEGF induces eNOS activation and increases NO production in endothelial cells via the Akt-dependent phosphorylation of Ser-1177 eNOS.37 Recently, it has been reported that AT2 receptor stimulation leads to inactivation of Akt by decreasing the phosphorylation of Akt at Ser-473.28 We therefore hypothesized that the AT2 receptor interferes with VEGF signaling on the level of Akt activation and studied the influence of AT2 receptor activation on the phosphorylation status of the VEGF receptor, Akt and eNOS. In accordance with the proposed model, the stimulation of AT2 receptors decreased the VEGF-induced phosphorylation of Akt and its downstream effector eNOS. As the autophosphorylation of Flk-1/KDR as well as its expression was unaffected, an interference on the level of the VEGF receptor activation or expression can be excluded. Therefore, we suggest that VEGF-antagonistic effects of Ang II on endothelial cell migration und in vitro tube formation are mediated by the AT2 receptor–induced reduction of VEGF-induced Akt activation.

It has recently been reported that AT2 receptor–mediated inactivation of Akt may exert proapoptotic actions.28 In addition, it is well established that VEGF promotes endothelial cell survival via Flk-1 receptor–mediated Akt activation.38 Thus, another explanation for the inhibition of cell migration and tube formation might be the induction of endothelial cell apoptosis by AT2 receptors.25 Although we were able to confirm the antiapoptotic action of VEGF on endothelial cells, an induction of apoptosis by Ang II in the experimental settings used for cell migration and tube formation assays was not detectable. Although we cannot exclude that the induction of apoptosis by AT2 receptors may become relevant after prolonged incubation, it is apparently not responsible for the inhibitory effect of Ang II on VEGF-induced endothelial cell migration and in vitro tube formation.

In the present study, Ang II alone induced endothelial cell migration only in EA.hy926 cells at high concentrations (10-5 mol/L). This induction was apparently mediated through AT1 receptor signaling as it could be blocked by losartan. Furthermore, the AT2 receptor antagonist PD123,319 significantly reduced the minimum effective concentration of Ang II from 10-5 to 10-8 mol/L. This strongly suggests antimotogenic and AT1 receptor–antagonistic actions mediated by the AT2 receptor. In contrast, Ang II in presence or absence of Ang II receptor antagonists had no effect on basal migration or tube formation of primary endothelial cells. These results are in line with the findings of Bell and Madri14 who showed that exogenous Ang II did not affect basal bovine aortic endothelial cell migration. Furthermore, Ang II did not alter the in vitro tube formation of BRECs.34 Thus, our results reveal a difference between primary endothelial cells (HDMECs/HCAECs) and EA.hy926 cells concerning the motogenic responsiveness to Ang II. The permanent cell line EA.hy926 was obtained by the fusion of human umbilical vein endothelial cells with a human lung carcinoma cell line (A549) and possesses "differentiated" endothelial characteristics, eg, the continued expression of eNOS and factor VIII-related antigen.21 Nonetheless the hybridization may result in a loss or transformation of endothelial cell characteristics which in turn may account for differences in motogenic behavior of EA.hy926 cells. However, we also noted that EA.hy926 cells express higher amounts of the AT1 receptor than the primary endothelial cells. Thus, the stimulation of basal migration in EA.hy926 cells may simply result from the more pronounced signaling of the AT1 receptors.

The AT2 receptor possesses a 7-transmembrane domain structure, which is common to all G protein–coupled receptors.39 However, data demonstrating an interaction of this receptor with G proteins are rare. AbdAlla et al19 recently reported that the AT2 receptor acts as an AT1 receptor antagonist by dimerization with the AT1 receptor. This antagonism did not require activation of the AT2 receptor nor involved activation of a G protein. This mechanism is apparently not relevant to the antimotogenic effect of AT2 receptors on endothelial cells. In our study the AT2 receptor–mediated inhibitory effect could be blocked by the AT2 receptor antagonist PD123,319 and was absent in endothelial cells treated with PTX. In addition, the Ang II–induced decrease of VEGF-stimulated Akt and eNOS phosphorylation was blunted in PTX-treated endothelial cells. These data are in agreement with our hypothesis that AT2 receptor activation interferes with VEGF-induced endothelial cell migration at the level of Akt phosphorylation. They further indicate that Ang II–mediated activation of the AT2 receptor as well as productive coupling to a Gi/o protein is required for these effects. In accordance with our results the AT2 receptor obviously couples to G{alpha}i1/-2 in rat fetal tissue.40 More recently, Lehtonen et al25 showed that AT2 receptor–mediated apoptosis in PC-12W cells is prevented by PTX. Hence, we can provide further evidence for the important role of productive coupling of the AT2 receptor to Gi/o-mediated signaling.

Intriguingly, treatment of endothelial cells with PTX for 24 hours was associated with a concentration-dependent reduction of endothelial cell migration. We could not detect an increase in number of apoptotic cells by PTX treatment, and on visual inspection, no signs of cellular impairment were detectable. Thus, the effect is most likely not due to enhanced cell death. Nevertheless, the important role of receptors coupling to PTX-sensitive G proteins for cell migration has previously been demonstrated. PTX treatment effectively impairs migration of the bladder carcinoma-derived cell line J82 and migration of leukocytes toward different chemotactic stimuli.41,42 Thus, PTX might be able to block a motogenic stimulus secreted by the endothelial cells themselves. For example, platelet activating factor is produced by vascular endothelial cells, directly stimulates cell migration, and apparently signals through a PTX-sensitive pathway.43,44

In conclusion, our study demonstrates a pertussis toxin–sensitive inhibitory effect of the AT2 receptor on VEGF-induced migration of endothelial cells, which involves a decrease in the phosphorylation of Akt and eNOS. Furthermore, AT2 receptor stimulation impaired VEGF-induced tube formation of HDMECs. Taken together, our findings uncover mechanisms by which the AT2 receptor may exert antiangiogenic actions that require Gi/o protein activation.


*    Footnotes
 
Original received November 13, 2002; resubmission received July 2, 2003; accepted July 16, 2003.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
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 up arrowDiscussion
 *References
 

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