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(Circulation Research. 2000;86:892.)
© 2000 American Heart Association, Inc.

Cellular Biology

Vascular Endothelial Growth Factor–Stimulated Actin Reorganization and Migration of Endothelial Cells Is Regulated via the Serine/Threonine Kinase Akt

Manuel Morales-Ruiz, David Fulton, Grzegorz Sowa, Lucia R. Languino, Yasushi Fujio, Kenneth Walsh, William C. Sessa

From the Departments of Pharmacology (M.M.-R., D.F., G.S., W.C.S.) and Pathology (L.R.L.) and Molecular Cardiobiology Program (M.M.-R., D.F., G.S., W.C.S.), Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Conn, and Division of Cardiovascular Research (Y.F., K.W.), St Elizabeth’s Medical Center, Boston, Mass.

Correspondence to William C. Sessa, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Ave, New Haven, CT 06536-0812. E-mail william.sessa{at}yale.edu

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Abstract—Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding ß-galactosidase (ß-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with ß-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of ß-gal–infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.

Key Words: vascular endothelial growth factor • angiogenesis • cell migration • nitric oxide • actin

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Phosphoinositide 3-kinases (PI[3] kinases) constitute a multifunctional family of enzymes that are activated by receptor tyrosine kinases such as the insulin, platelet-derived growth factor, and vascular endothelial growth factor (VEGF) receptors.¹ Ligand-dependent recruitment of PI(3) kinase subunits induces the phosphorylation of the inositol ring of phosphatidylinositol (PtdIns) lipids at the D-3 position producing the second messengers PtdIns(3,4)P₂ and PtdIns(3,4,5,)P₃. These inositol lipids are implicated in numerous cellular functions, including signal transduction to the nucleus,² membrane trafficking,³ cytoskeletal rearrangement, and cell migration.^{4 5 6} In the context of cell migration, inhibitors of PI(3) kinase, as well as dominant-negative suppression of the enzyme, prevent growth factor–induced cytoskeletal changes and migration. However, the downstream effectors of PI(3) kinase responsible for these effects are not well established.

One downstream effector of PI(3) kinase is the serine/threonine kinase Akt (or protein kinase B).⁷ Upon receptor activation, Akt is recruited to the plasma membrane and binds to inositol lipids via its pleckstrin homology domain. Once in the membrane, Akt is phosphorylated by phosphoinositide-dependent kinases, and phosphorylation enhances its catalytic activity toward a variety of diverse substrates.⁸ Akt is an important regulator of various cellular processes, including metabolism and cell survival.⁹ Recently, we and others have shown that Akt can phosphorylate bovine endothelial NO synthase (eNOS) on serine 1179 (or serine 1177 in the human ortholog), resulting in eNOS activation and NO production.^{10 11 12} These findings, in addition to reports demonstrating a role for NO in endothelial cell migration promoted by growth factors such as endothelin and VEGF,^{13 14 15} suggested that the Akt/eNOS pathway functions to regulate endothelial cell migration. Therefore, we undertook the present study to examine whether the Akt/eNOS pathway participates in VEGF-induced endothelial cell migration, a necessary component of the angiogenic response.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cell Culture and Reagents
Bovine lung microvascular endothelial cells (BLMVECs, Vec Technologies) and bovine aortic endothelial cells were cultured as described previously.^{16 17 18}

Adenoviral Constructs
ß-Galactosidase (ß-gal), hemagglutinin (HA)-tagged inactive phosphorylation mutant Akt (AA-Akt), and carboxyl-terminal HA-tagged constitutively active Akt (myr-Akt) were generated as described previously.^{10 19} BLMVECs were infected with 100 multiplicity of infection of adenovirus containing ß-gal, AA-Akt, or myr-Akt for 12 hours. The virus was removed, and cells were left to recover for 12 hours in complete medium. These conditions resulted in uniform expression of the transgenes in close to 100% of the cells (determined by infection with ß-gal followed by staining for ß-gal activity) and equal expression of Akt proteins, based on Western blotting as described.¹⁰

Cell Migration Assay
Migration assays were performed as described previously using a Boyden chamber (Neuroprobe).²⁰ BLMVECs were infected with adenoviruses for ß-gal. AA-Akt or myr-Akt, as described above, were serum starved overnight and detached using trypsin (0.05% vol/vol)/EDTA (0.53 mmol/L). Approximately 20 000 cells were suspended in M199 containing BSA (0.1%) and were added to the lower chamber. Polycarbonate filters (8-µm pores; Poretics Corp) were coated with 100 µg/mL type I collagen (Collaborative Biomedical Products). The top half of the chamber was attached, and the chamber was incubated in an inverted position at 37°C for 2 hours to allow uniform cell attachment to the filter. VEGF (1 to 100 ng/mL), N^G-nitro-L-arginine methyl ester (L-NAME; 3 mmol/L), N^G-nitro-D-arginine methyl ester (D-NAME; 3 mmol/L), or vehicle (M199 with 0.1% BSA) was added to the lower chamber. The chamber was incubated for an additional 5 hours at 37°C. After incubation, cells were fixed with ethanol (70%), and nonmigrating cells on the upper surface of the filter were removed. Migrated cells were stained with Giemsa and counted (at 400x magnification) in 3 random fields per well. Each experiment was performed in triplicate, and migration was expressed as the number of total cells counted per well. In some experiments, BLMVECs were preincubated with or without L-NAME (3 mmol/L), D-NAME (3 mmol/L), LY294002 (10 µmol/L), or wortmannin (100 nmol/L) for 30 minutes in M199 with 0.1% BSA at 37°C. In preliminary experiments, this concentration of L-NAME, but not D-NAME, completely blocked VEGF or calcium ionophore–stimulated NO production or cGMP accumulation in a reporter bioassay system as described.^{18 21 22} In addition, the concentrations of both LY294002 and wortmannin completely abolished VEGF- or serum-stimulated Akt phosphorylation.

Adhesion Assay
Cell adhesion was assayed in 96-well plates precoated with type I collagen (1, 3, 10, and 30 µg/mL) overnight, as previously described²³ (see also online-only expanded Materials and Methods; http://www.circresaha.org).

Measurement of NO Release
For measurement of NO, the release of NO₂^-, the stable breakdown product of NO in aqueous medium, was determined as previously described¹⁸ (see also online-only expanded Materials and Methods; http://www.circresaha.org).

Confocal Fluorescence Microscopy
BLMVECs infected with adenoviruses for ß-gal, AA-Akt, or myr-Akt were plated on gelatin-coated 35-mm plates (MatTek) as previously described²¹ (see also online-only expanded Materials and Methods; http://www.circresaha.org).

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Previously, we have shown that 2 distinct inhibitors of PI(3) kinase, LY294002 and wortmannin, attenuated VEGF-stimulated NO release from endothelial cells.²⁴ To examine whether these inhibitors affect endothelial cell migration, BLMVECs were pretreated with these compounds and placed into a Boyden chamber migration assay in the absence or presence of VEGF (10 ng/mL) as the chemoattractant. Treatment with either LY294002 (10 µmol/L) or wortmannin (100 nmol/L) did not influence basal endothelial cell migration in the absence of VEGF (19.2±1.7, 18.3±2.0, and 12.3±2.1 migrated cells per field for cells treated with vehicle, LY294002, and wortmannin, respectively) but attenuated VEGF-stimulated migration (33.2±2.6, 23.8±1.7 [P<0.05], and 19.7±1.5 [P<0.05] migrated cells per field for cells treated with vehicle, LY294002, and wortamannin, respectively, in the presence of VEGF; n=3). To examine the putative role of Akt as a downstream effector of PI(3) kinase in endothelial cell migration, BLMVECs were infected with adenoviruses for ß-gal, AA-Akt, or myr-Akt and subjected to a cell migration assay in the absence or presence of a chemotactic gradient toward VEGF (Figure 1A). Before the cell migration experiments, we documented the expression of the transgenes (Figure 1A, inset). AA-Akt and myr-Akt were expressed to the same extent in infected BLMVECs. Next, we documented the effects of the adenoviruses on VEGF-stimulated NO release. VEGF stimulated NO release from ß-gal–infected cells (from 0.47±0.08 to 0.95±0.1 pmol/µg protein), whereas AA-Akt infection of BLMVECs attenuated VEGF-driven NO production (from 0.67±0.04 to 0.7±0.1 pmol/µg protein). myr-Akt transduction increased basal NO release by itself, an effect augmented by the addition of VEGF (from 2.8±0.01 to 3.6±0.56 pmol/µg protein; n=3 experiments). These results are similar to our previously published findings.¹⁰ Next, we examined VEGF-induced cell migration. VEGF dose-dependently increased endothelial cell migration in cells transduced with ß-gal virus (Figure 1A). VEGF-induced migration was identical in ß-gal–infected and noninfected cells (not shown). Transduction of BLMVECs with myr-Akt dramatically increased basal endothelial cell migration compared with endothelial cells infected with ß-gal or AA-Akt (0 point on Figure 1A), and VEGF further increased migration (at 10 ng/mL) in myr-Akt–transduced cells. In contrast, VEGF-stimulated cell migration was markedly attenuated by infection with adenoviral AA-Akt. Identical results were obtained in endothelial cells of systemic origin (ie, bovine aortic endothelial cells; see Figure 1 online [http://www.circresaha.org]). These results demonstrate that activation of Akt is necessary to stimulate endothelial cell migration in response to VEGF, whereas constitutive activation of Akt is sufficient to promote migration in the absence of a chemotactic agent.

View larger version (28K): [in this window] [in a new window]   Figure 1. VEGF-induced endothelial cell migration is potentiated by constitutively active Akt and inhibited by activation-deficient Akt. A, BLMVECs were infected with adenoviruses for ß-gal, AA-Akt, and myr-Akt and subjected to a migration assay as described in Materials and Methods. Results are expressed as mean±SEM of 3 separate experiments performed in triplicate. *P<0.05 compared with ß-gal– or AA-Akt–infected cells; **P<0.05 compared with basal conditions. Inset, Expression of eNOS (with eNOS monoclonal antibody) and the corresponding Akt protein (AA-Akt or myr-Akt with HA monoclonal antibody) in these cells. B, Serum-starved BLMVECs infected with adenoviruses for ß-gal, AA-Akt, or myr-Akt were incubated in 96-well plates coated with collagen (10 µg/mL) and incubated at 37°C for 2.5 hours. Adherent cells were quantified as described. Data are mean±SEM of 1 experiment performed in triplicate.

Adhesion assays were performed to determine whether the effects of Akt on endothelial cell migration could be attributed to changes in cell adhesion to the collagen matrix. Figure 1B shows that BLMVECs transduced with ß-gal, AA-Akt, or myr-Akt adhered similarly to collagen. Thus, the ability of the Akt transgenes to influence cell migration were not due to effects on overall cell adhesion.

VEGF-stimulated endothelial cell proliferation, migration, and angiogenesis can be blocked by L-arginine–substituted analogues that inhibit NO synthase (NOS),^{14 24} and VEGF-induced angiogenesis is markedly attenuated in eNOS knockout mice.²⁵ Accordingly, we examined the effects of the NOS inhibitor, L-NAME, or the inactive D isomer, D-NAME, on basal and VEGF-stimulated cell migration in endothelial cells infected with adenoviruses for ß-gal or myr-Akt. In endothelial cells transduced with ß-gal (Figure 2A), VEGF stimulated cell migration, an effect blocked by L-NAME but not by D-NAME. L-NAME had no effect on basal migration, suggesting that VEGF activation of NOS and the subsequent production of NO is involved in cell migration. In contrast, myr-Akt markedly stimulated basal endothelial cell migration, an effect that was not influenced by L-NAME. Under these conditions, the NOS inhibitor reduced myr-Akt–stimulated NO₂^– by >97% (n=3). However, VEGF-stimulated cell migration in myr-Akt–transduced BLMVECs was blocked by L-NAME but not by D-NAME (Figure 2B). These results suggest that the Akt-NOS pathway is necessary for VEGF-induced cell migration and that myr-Akt, while causing NO release, stimulates cell migration in a NOS-independent manner.

View larger version (14K): [in this window] [in a new window]   Figure 2. Inhibition of NO production blocks VEGF-induced cell migration in BLMVECs transduced with ß-gal and myr-Akt but does not inhibit myr-Akt–induced basal cell migration. A, BLMVECs were infected with an adenovirus for ß-gal and were subjected to a migration assay in the absence or presence of VEGF (10 ng/mL) or of L-NAME or D-NAME (3 mmol/L of each) added in the upper chamber. At the end of the treatment, the number of migrated cells was counted as above. Results are expressed as mean±SEM of 2 separate experiments performed in triplicate. *P<0.01 compared with migration of basal cells, L-NAME–treated cells, and L-NAME– and VEGF–treated cells. B, BLMVECs infected with adenovirus for myr-Akt were subjected to a migration assay as described above. *P<0.05 compared with migration of basal cells, L-NAME–treated cells, and L-NAME– and VEGF–treated cells.

Cell migration is associated with regulation of the actin cytoskeleton. As shown previously, VEGF induces edge ruffling²² and stress fiber formation in cultured endothelial cells.²⁶ In quiescent BLMVECs infected with ß-gal, F-actin was found mostly in membrane structures and unorganized fibers throughout the cell (Figure 3, left panel, top). As expected, treatment with VEGF induced the formation of long, condensed stress fibers (Figure 3, right panel, top). To explore the possibility that VEGF signals through Akt lead to stress fiber formation, BLMVECs were transduced with AA-Akt and myr-Akt, and the effects of VEGF were examined. As seen in control cells infected with AA-Akt, there was less structured F-actin compared with cells infected with ß-gal (Figure 3, left panel, middle). Importantly, VEGF-stimulated stress fiber formation was markedly attenuated in cells expressing AA-Akt (Figure 3, right panel, middle). Furthermore, infection of BLMVECs with adenoviral myr-Akt induces stress fiber formation and reorganization of F-actin (Figure 3, left panel, bottom). Because of the profound effect of myr-Akt by itself on F-actin, it was difficult to visualize VEGF-stimulated rearrangement of the actin cytoskeleton (Figure 3, right panel, bottom). These data indicate that VEGF-induced cell migration and F-actin rearrangement are dependent on Akt and that constitutively activated Akt is sufficient to cause cell migration most likely because of its effects on stress fiber formation.

View larger version (99K): [in this window] [in a new window]   Figure 3. Akt-dependent stress fiber formation induced by VEGF. BLMVECs were infected with adenoviruses for ß-gal (top), AA-Akt (middle), or myr-Akt (bottom) incubated with PBS (control) or with VEGF (50 ng/mL for 5 minutes). F-Actin was detected in fixed and permeabilized cells using Texas Red–labeled phalloidin. Representative fields are shown. In ß-gal– and myr-Akt–infected cells, solid arrows denote stress fibers. In the AA-Akt–infected cells, open arrows reflect the lack of stress fiber formation. Similar results were obtained in 2 additional experiments. Bar=10 µm.

To our knowledge, these results are the first to demonstrate a critical role for Akt in the migration of mammalian cells. Our findings demonstrating that Akt is important for endothelial cell migration are supported by various studies demonstrating a role of PI(3) kinase in cell migration^{4 5} and by a recent study in Dictyostelium showing that Akt is required for migration of cells toward the chemoattractant cAMP.²⁷

In the present study, VEGF-induced Akt activation, NO production, stress fiber formation, and migration appear to lie in a common pathway, given that activation-deficient Akt attenuates all of these responses. The importance of the VEGF/Akt/eNOS pathway is supported by the observations that inhibition of NOS blocks VEGF-driven NO release, endothelial cell migration, formation of endothelial tubelike structures in vitro, and angiogenesis in vivo.^{14 23 28 29} In the context of cell motility, the effectors of NO are not known; however, NO can influence the tractional forces in activated endothelial cells and influence remodeling of focal adhesions, perhaps by influencing tyrosine phosphorylation of focal adhesion kinase.¹⁵ The link between eNOS, Akt, and signaling through the small G protein Rho as a primary mechanism leading to stress fiber formation during cell migration is not known and is presently being explored. In addition, NO may modulate the activation of the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase/Hsp27 pathway that is crucial for VEGF-induced endothelial cell chemotaxis.²⁶

However, Akt activation of eNOS via phosphorylation and NO release is necessary for physiological migration in response to VEGF but is not sufficient for cell migration, based on our data with constitutively active Akt. Transduction of BLMVECs with myr-Akt markedly stimulated cell migration and profoundly affected cytoskeletal structure in the absence of VEGF. Surprisingly, inhibition of eNOS by L-NAME at concentrations that effectively block NO release^{18 21 25} had no influence on myr-Akt–stimulated cell migration. These observations present a paradox. On the one hand, both Akt activation and NO production are essential for the physiological migratory response to VEGF, yet on the other hand, NO is dispensable for migration induced by myr-Akt. In the context of constitutive activation, Akt must trigger separate but interacting pathways that lead to cell migration. Presumably, the persistent plasma membrane localization of myr-Akt, resembling the N-myristoylated oncogenic variant v-Akt,^{30 31} results in the unregulated, sustained activation of ancillary signaling pathway different from those activated by the transient association of cellular Akt with the plasma membrane.^{32 33}

In summary, VEGF can stimulate eNOS-derived NO production that is physiologically linked to cell migration. Upon activation of the VEGF receptor, activation of PI(3) kinase (pathway A in Figure 4) results in the PI(3) kinase–dependent phosphorylation of Akt, resulting in the phosphorylation of eNOS on serine 1179. Because AMP-activated protein kinase can also phosphorylate eNOS on the same residue, it is feasible that activation of this pathway by metabolic stress may trigger eNOS activation and cell migration.³⁴ Concomitantly, VEGF receptor engagement stimulates c-Src–dependent activation of phospholipase C- (PLC-)³⁵ (pathway B in Figure 4), resulting in an increase in cytoplasmic calcium. The increase in calcium activates calmodulin, thus enhancing the activity of phosphorylated eNOS. Paradoxically, myr-Akt, in the absence of VEGF, triggers NO release and cell migration; however, NO does not participate in the migratory response, because L-NAME at concentrations that effectively block NO release does not influence myr-Akt–driven migration (pathway C in Figure 4). Thus, this study highlights the physiological importance of the Akt/NOS pathway for VEGF-induced endothelial cell migration and demonstrates a novel function for Akt in controlling cell migration. Exploitation of this mechanism by selective inhibition of eNOS or Akt may provide a rationale for antiangiogenic therapy in the treatment of solid tumors.

View larger version (32K): [in this window] [in a new window]   Figure 4. Model of VEGF-stimulated Akt and NO release coupling to cell migration. Diagram illustrates VEGF signal transduction pathways that couple to eNOS and cell migration, including direct activation of PI(3) kinase and subsequent activation of Akt that phosphorylates eNOS, resulting in NO production (pathway A). In addition, VEGF can activate PLC- that increases intracellular calcium responsible for eNOS activation and NO production (pathway B). Finally, activation of Akt and eNOS by myr-Akt leads to uncoupled endothelial cell migration from NO release (pathway C). Broken lines indicate inhibition of pathways by AA-Akt or L-NAME.

	Acknowledgments

 
This work is supported by grants from the NIH (RO1HL51948, RO1HL50974, and RO1HL64793 to W.C.S.; T32HL10183 to D.F.; and RO1CA71870 to L.R.L.), a Grant-in-Aid from the American Heart Association (National Grant to W.C.S.), and grants from the Ministerio de Educación y Cultura (EX99-38446345) and from the Asociación Española para el Estudio del Hígado, Spain (to M.M.-R.). W.C.S. is an Established Investigator of the American Heart Association. We thank Genentech for the generous supply of VEGF.

Received December 8, 1999; accepted February 16, 2000.

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