Circulation Research. 2002;90:1243-1250
doi: 10.1161/01.RES.0000022200.71892.9F
(Circulation Research. 2002;90:1243.)
© 2002 American Heart Association, Inc.
Role of Akt Signaling in Vascular Homeostasis and Angiogenesis
Ichiro Shiojima,
Kenneth Walsh
From the Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Mass.
Correspondence to Kenneth Walsh, PhD, Molecular Cardiology/CVI, Boston University School of Medicine, 715 Albany St, W611, Boston, MA 02118. E-mail kwalsh{at}world.std.com
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Abstract
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Akt is a serine/threonine protein kinase that is activated by
a number of growth factors and cytokines in a phosphatidylinositol-3
kinase–dependent manner. Although antiapoptotic activity
of Akt is well known, it also regulates other aspects of cellular
functions, including migration, glucose metabolism, and protein
synthesis. In this review, Akt signaling in endothelial cells
and its critical roles in the regulation of vascular homeostasis
and angiogenesis will be discussed.
Key Words: Akt • endothelial cells • angiogenesis • statins • endothelial nitric oxide synthase
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Introduction
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Since the identification of several classes of receptor tyrosine
kinases and their ligands as crucial mediators of vascular development,
considerable progress has been made toward understanding the
process of angiogenesis at sites of tissue growth and/or repair.
1,2 A number of clinical trials are currently evaluating angiogenic
ligands for their ability to induce neovascularization in ischemic
tissues,
3,4 and the intracellular signaling pathways that mediate
the proangiogenic effects of these growth factors are being
extensively investigated. This review specifically focuses on
the role of phosphatidylinositol-3 kinase (PI3K)–Akt signaling
axis in endothelial cells because it is activated by many angiogenic
growth factors and it regulates downstream target molecules
that are potentially involved in blood vessel growth and homeostasis.
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PI3K-Akt Signaling Axis: Upstream Activators and Downstream Targets
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Akt was originally identified as a cellular counterpart of the
oncogene derived from murine AKT8 retrovirus.
5,6 The same gene
product was independently isolated as a protein kinase related
to protein kinase A and C and was therefore named as protein
kinase B (PKB) or RAC (related to protein kinase A and C).
7,8 Mammalian genomes contain three
Akt genes,
Akt1/PKB
,
Akt2/PKBß,
and
Akt3/PKB
, whereas
Drosophila melanogaster and
Caenorhabditis elegans contain one and two
Akt genes, respectively.
9,10 These
genes encode proteins containing a pleckstrin homology (PH)
domain in the amino terminus, a central kinase domain, and a
carboxy terminal regulatory domain. All 3 mammalian
Akt genes
are widely expressed in various tissues but Akt1 is most abundant
in brain, heart, and lung, whereas
Akt2 is predominantly expressed
in skeletal muscle and embryonic brown fat, and
Akt3 is predominantly
expressed in brain, kidney, and embryonic heart.
7,11–13 In unstimulated cells, Akt protein exists in cytoplasm and the
two regulatory phosphorylation sites at threonine at 308 and
serine at 473 are in an unphosphorylated state. On growth factor
stimulation, the PH domain binds to the lipid products of PI3K,
and Akt is recruited to plasma membrane. Akt is then sequentially
phosphorylated at T308 and S473 by upstream kinases referred
to as 3-phosphoinositide–dependent protein kinase 1 (PDK1)
and PDK2, respectively, to yield a fully activated kinase (
Figure 1).
14,15 PDK1 has been isolated and characterized,
15 but the
identity of PDK2 is still controversial. Several candidate molecules
have been suggested to be a potential S473-kinase including
integrin-linked kinase (ILK), MAP kinase–activated protein
kinase 2 (MK2), PDK1 (conversion of substrate specificity in
association with protein kinase C–related kinase-2 [PRK2])
and Akt itself (autophosphorylation).
16–19 Fully activated
Akt becomes available to phosphorylate its downstream substrates
and a portion of these molecules detach from the plasma membrane
and translocate to various subcellular locations including nucleus.
20 Akt is then dephosphorylated and inactivated by protein phosphatases
such as protein phosphatase 2A (PP2A).
21
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Figure 1. Mechanism of Akt activation and partial list of downstream molecules. Akt is activated by growth factors or cytokines in a PI3K-dependent manner, and phosphorylation of two residues by PDK1 (T308) and PDK2 (S473) is required for its full activation. Downstream target molecules are grouped according to their function. Note that these downstream molecules include both direct Akt substrates and indirect downstream effectors.
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Akt is a critical regulator of PI3K-mediated cell survival.22,23 A large number of studies have demonstrated in various cell types that constitutive activation of Akt signaling is sufficient to block cell death induced by a variety of apoptotic stimuli and that transduction of dominant-negative Akt inhibits growth factor–induced cell survival.24–26 The prosurvival function of Akt has also been demonstrated in the context of the intact organism. Mutation of Drosophila Akt leads to embryonic lethality due to massive apoptosis during embryogenesis,27 and Akt1 mutant mice exhibit increased spontaneous apoptosis in testis and thymus.28 Several downstream targets of Akt are recognized to be apoptosis-regulatory molecules including Bad, FKHR family of forkhead transcription factors, and IKK,29–35 and these findings are consistent with the notion that Akt functions as a survival kinase. However, other downstream effectors of Akt are involved in different aspects of cellular regulation. For example, (1) Akt enhances glucose uptake by inducing membrane translocation of the glucose transporter GLUT4,36 (2) Akt promotes glycogen synthesis through the phosphorylation and inactivation of glycogen synthase kinase-3 (GSK-3),37 (3) Akt regulates cell cycle and cellular senescence, at least in part, through modulating the activities of E2F, p21, MDM2, and human telomerase reverse transcriptase subunit (hTERT),38–44 and (4) Akt enhances protein synthesis through increasing the phosphorylation of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and 70-kDa S6 kinase (S6K1),15,45 although S6K1 may also be directly activated by PDK1 in a PI3K-dependent and Akt-independent fashion (Figure 1).46,47 Taken together, it is more appropriate to classify Akt as a multifunctional protein kinase rather than a simple regulator of cell survival.
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Akt-Dependent Survival Signals in Endothelial Cells
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Although originally identified as a factor that induces vascular
permeability, vascular endothelial growth factor (VEGF) exhibits
multiple biological activities in endothelial cells, including
the enhancement of endothelial cell survival.
48 VEGF effects
on cell survival have been shown to be mediated by Flk1/VEGFR2-PI3K-Akt
pathway.
49,50 Subsequently, it was also shown that several other
endothelial cell stimuli including angiopoietin-1 (Ang-1),
51,52 insulin,
53 insulin-like growth factor-I (IGF-I),
54 sphingosine-1-phosphate
(S1P),
55,56 hepatocyte growth factor,
57 the small proteoglycan
decorin,
58 fluid shear stress,
59 estrogen,
59a reactive oxygen
species,
59b and corticosteroids
59c also activate PI3K-Akt signaling,
illustrating the central role of this pathway in controlling
endothelial cell viability.
Growth factor activation of angiogenesis is dependent on proper endothelial cell–extracellular matrix attachment,60 and in the absence of matrix attachment, cells undergo apoptotic cell death through a process termed anoikis (a Greek word for "homelessness").61 VEGF activation of Akt in endothelial cells is dependent on matrix attachment, and constitutively active Akt blocks cell detachment–induced apoptosis.50 These findings suggest that matrix attachment is required for growth factors to activate Akt and maintain endothelial cell viability. Cell attachment is mediated mainly through the engagement of extracellular matrix with integrin molecules. When integrins bind to extracellular matrix they become clustered and associate with the actin cytoskeleton through adaptor/signaling molecules, which further promotes integrin clustering and the assembly of actin filaments and leads to the formation of focal adhesion and activation of intracellular signaling.62 The v integrin combinations have been most extensively investigated in terms of their roles in angiogenesis.63 Endothelial cells stimulated with angiogenic growth factors or those in newly formed vessels express high levels of vß3 integrin, and antagonists against vß3 or vß5 integrin block the growth factor-induced angiogenesis. It has also been shown that vß3 integrin associates with VEGF and platelet-derived growth factor (PDGF) receptors and potentiates VEGF or PDGF signaling, respectively.63 Because several integrin signaling molecules including focal adhesion kinase (FAK), ILK, and Shc have been implicated in Akt activation,61 downregulation of Akt activity induced by cell detachment is likely due to the decrease in integrin-dependent Akt activation. Caspase-mediated cleavage of Akt is also implicated in the downregulation of Akt protein level during long-term suspension culture.64 Collectively, these findings suggest that integrin signaling induced by cell attachment (outside-in signal) is an important regulator of growth factor–dependent endothelial cell survival and angiogenesis through PI3K-Akt pathways. Furthermore, VEGF-induction of inside-out signals has also been shown to activate integrins, 65 suggesting that integrin and growth factor signaling are cooperative and synergistic with regard to activation of Akt signaling (Figure 2).
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Figure 2. Growth factor- and cell attachment-dependent survival is mediated by PI3K/Akt signaling in endothelial cells. Integrin-dependent signals are prerequisite for growth factor–mediated activation of Akt. Akt-mediated crosstalk between these two signaling systems synergistically promotes endothelial cell survival.
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Currently, relatively little is known about the downstream mediators of Akt-dependent survival pathway in endothelial cells, although several candidate molecules has been identified including survivin,51 FLICE-inhibitory protein (FLIP),66 and MEKK3.67 Thus, possible combinations of these and other unidentified Akt target molecules may control endothelial cell survival depending on the context of pro- and antiapoptotic stimuli encountered in the cellular environment.
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Regulation of Endothelial Nitric Oxide Synthase (eNOS) Activity by Akt
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In addition to its antiapoptotic effects, VEGF induces hypotension
in the intact organism, nitric oxide (NO)–dependent vasodilation
in isolated coronary arteries, and NO release in isolated vessels
and in cultured endothelial cells.
68–70 Early studies
demonstrated that VEGF-induced increase in NO release from endothelial
cells is attenuated by PI3K inhibitors,
71 and subsequently,
it was demonstrated that VEGF stimulates Akt-mediated eNOS phosphorylation
at Ser1177 (in human eNOS, equivalent to Ser1179 in bovine eNOS),
leading to an increase in eNOS activity.
72,73 It is also reported
that production of NO in response to fluid shear stress in cultured
endothelial cells is controlled by Akt-dependent phosphorylation
of eNOS,
73 although another study has shown that shear stress
induces eNOS phosphorylation predominantly through a protein
kinase A–dependent, Akt-independent mechanism.
74 Studies
in intact animals have shown that overexpression of constitutively
active Akt in the vascular endothelium increases resting diameter
and blood flow, whereas transduction of dominant-negative Akt
attenuates endothelium-dependent vasodilation induced by acetylcholine,
75,75a demonstrating that Akt functions as a regulator of vasomotor
tone in vivo. PI3K/Akt signaling has also been implicated in
the control of endothelium-dependent vasorelaxation induced
by adrenomedullin,
76 and hyperglycemia has been shown to lead
to the glycosylation of the Akt phosphorylation site in eNOS,
resulting in an inhibition of eNOS activity.
77
The activity of eNOS is also regulated by subcellular localization and/or protein-protein interactions. Of note, eNOS has been shown to be localized in a specific domain of plasma membrane called caveolae and to interact with caveolin-1 through caveolin-1 scaffolding domain, which inhibits eNOS activity.78–81 Although originally implicated in transmembrane trafficking of macromolecules, the finding that caveolae contain a variety of signaling molecules and caveolin-1 directly interacts with those caveolae-associated proteins have suggested that caveolae and caveolin-1 are involved in the compartmentalization and integration of signal transduction pathways at the cell membrane. Consistent with the inhibitory role of caveolin-1 on eNOS activity, administration of caveolin-1 scaffolding domain fused to cellular internalization sequences in vivo attenuates eNOS activity,82 and acetylcholine-induced vasorelaxation and NO production are enhanced in caveolin-1-deficient mice.83 The targeting of eNOS to caveolae, however, seems to be required for efficient and proper activation of eNOS on stimulation, because conditions that inhibit the localization of eNOS in caveolae also attenuate eNOS activity.84,85 It has also been shown that eNOS interacts with heat shock protein 90 (Hsp90) on stimulation with VEGF or shear stress, and this interaction enhances eNOS activity.86 Interestingly, Akt also interacts with Hsp90 on stimulation and this interaction enhances Akt enzymatic activity,87 suggesting that Hsp90 may serve as a scaffold protein for the efficient phosphorylation of eNOS by Akt at caveolae (Figure 3).88,89,89a
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Figure 3. Schematic illustration of the Akt-eNOS interaction at caveolae. Caveolin-1 is localized to caveolae and associates with a number of regulatory molecules including eNOS. Association of eNOS with caveolin-1 negatively regulates eNOS activity, although targeting of eNOS to caveolae is required for proper eNOS function. Activated Akt and eNOS also associate with Hsp90. Hsp90 is believed to function as a scaffold protein for activation of eNOS by Akt-mediated phosphorylation.
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Regulation of Endothelial Cell Migration by Akt
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The ability of endothelial cells to migrate and form capillary-like
structures is essential for angiogenesis in vivo.
1 VEGF enhances
endothelial cell migration and capillary-like structure formation
in vitro and these activities of VEGF are PI3K-Akt–dependent.
90–92 S1P has also been shown to enhance endothelial cell migration
and capillary formation in vitro through the activation of the
endothelial differentiation gene (EDG) family of G protein–coupled
receptors and PI3K-Akt–dependent pathways.
56,93,94 Conversely,
oxidized LDL inhibits endothelial cell migration toward VEGF
by promoting the dephosphorylation of Akt.
95
Studies in other cell types have also implicated PI3K and Akt in the control of directional cell migration and the sensing of chemoattractant gradients by the cell. It has been shown that Akt transiently localizes to the leading edge membrane of migratory cells in a PI3K-dependent manner,96,97 and gene ablation studies in mice have demonstrated that PI3K is required for chemotaxis and chemoattractant-dependent activation of Akt in macrophages and neutrophils.98–100 Akt has been shown to be required for chemotaxis in Dictyostelium cells as well.96
Cellular movement requires the reorganization of actin cytoskeleton and distinct patterns of actin reorganization are required as cells establish leading edge and then generate contractile force to migrate forward.101 Previous studies have implicated the Rho family of small G proteins as one of the major regulators of actin reorganization. Among Rho family members, Rho, Rac, and Cdc42 are most widely studied and each regulates specific aspects of cytoskeletal reorganization. Rho stimulates cytoplasmic stress fiber formation and actomyosin-based contractility, Rac induces membrane ruffling and extension of lamellipodia, and Cdc42 induces the extension of membrane protrusions (filopodia) and is also involved in chemoattractant gradient sensing.102,103 In endothelial cells, it has been shown that VEGF-induced cell migration is dependent on Rho family GTPases.104,105 However, the relationship between Akt and Rho family of G proteins is complicated and controversial. On one hand, Akt was shown to negatively regulate Rac1 activity by phosphorylating Rac1 and inhibiting its GTP-binding activity.106 In contrast, a recent study has demonstrated that Akt phosphorylates S1P receptor EDG-1 and induces Rac activation and cell migration in endothelial cells.107 Other reports show that Rac and Cdc42 are situated upstream of Akt and that they promote Akt signaling.108–110 Consistent with these findings, Akt has been shown to be required for cell motility induced by Rac or Cdc42 in fibroblasts.108
Another possible downstream effector of Akt that regulates cell motility is p21-activated protein kinase (PAK). PAK was originally identified as a Rac1-binding protein that specifically interacts with GTP-bound form of Rac.111 Subsequently, it was shown that PAK is activated by Rac or Cdc42 and that it regulates polarized cytoskeletal reorganization.111 Recently it was shown in Dictyostelium cells that Akt regulates cell polarity and chemotaxis through the regulatory phosphorylation of PAK,112 suggesting a direct functional link between Akt and PAK in the regulation of cytoskeletal reorganization. In mammalian fibroblasts, it was also shown that Akt stimulates PAK1 activation and dominant-negative Akt inhibits Ras-induced activation of PAK1.113 However, the Akt phosphorylation site in Dictyostelium PAK is not conserved in mammalian PAK1, suggesting an indirect activation of mammalian PAK1 by Akt. Nonetheless, PAK family of protein kinases are attractive candidates for Akt effectors in the regulation of endothelial cell migration, and may be a convergence point of signals from Rac/Cdc42 and Akt.
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Statins and Akt Signaling
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The 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase
inhibitors, or statins, are widely prescribed for the treatment
of hypercholesterolemia, and several clinical trials have demonstrated
that statins are effective for both primary and secondary prevention
of coronary artery diseases.
114 It has also been shown that
statins rapidly improve vasomotor responses of atherosclerotic
coronary arteries both in humans and in animal models,
115–117 and studies in normocholesterolemic animals revealed that statins
protect against stroke and myocardial ischemia/reperfusion injury
possibly through NO-dependent mechanisms.
118,119 These findings
are consistent with the notion that cardioprotective effects
of statins are partly independent of their serum lipid-lowering
effects and may be due to the activation of eNOS in vascular
endothelium.
Recent research has revealed a link between statins and Akt. Statins have been shown to rapidly promote the activation of Akt in endothelial cells leading to eNOS phosphorylation and increased NO production.91 Low statin concentrations have been shown to protect endothelial cells from serum deprivation–induced apoptosis and promote capillary-like structure formation on matrigel in an Akt-dependent manner, whereas higher concentrations are toxic.91 Consistent with their Akt-activating function, treatment with clinically-relevant doses of statins enhances angiogenesis in the ischemic hindlimbs of normocholesterolemic animals through an eNOS-dependent mechanism.91,120 It has also been shown that the activation of angiogenesis by statins is biphasic: low doses promote vessel formation, whereas high doses inhibit angiogenesis.121 More recently, it was shown that statins enhance the mobilization of endothelial progenitor cells (EPCs) from bone marrow to newly forming blood vessels in a PI3K-Akt–dependent manner,122,123 suggesting another mechanism of Akt-dependent proangiogenic effects of statins. Moreover, it has been shown that statins promote EPC mobilization in patients with stable coronary heart diseases.124 Although there are numerous lines of evidence to suggest that statins promote endothelial cell function and angiogenesis, there is no evidence in clinical studies linking statin treatment to increase in cancer risk.125
Activation of Akt by statins is blocked by treatment with wortmannin or LY294002,91 suggesting that statin activation of Akt is mediated by PI3K. However, the mechanisms by which statins activate PI3K are unknown at present. In this regard, statins have been shown to decrease caveolin-1–eNOS interaction and enhance the formation of eNOS-Hsp90-Akt complex in endothelial cells,89 although it is not clear whether these effects of statins are secondary to Akt activation or not. It should also be noted that endothelial cells are relatively unique in this response because activation of Akt by statins is not observed in cardiac or smooth muscle cells, 91 suggesting an endothelial cell–specific pathway of PI3K-Akt activation. A recent report has shown that low, clinically relevant doses of statin activate endothelial Ras and promote Akt and eNOS phosphorylation.126 It was also reported that higher statin doses are toxic to endothelial cells although they promote an increase in eNOS protein expression. Presumably, the toxicity results from an inhibition of protein prenylation,127 and this may explain the antiangiogenic effects observed in studies performed with higher statin concentrations.128,129
In addition to their proangiogenic effects, statins have also been shown to exhibit antithrombotic actions in humans, which appears to be independent of their serum cholesterol-lowering effects.130 Recent studies have shown that PI3K-Akt pathway inhibits the expression of tissue factor,131,132 which is the primary cellular initiator of blood coagulation and whose expression is induced in endothelial cells and macrophages by a number of stimuli, including interleukin-1ß and tumor necrosis factor-.133 Although VEGF activates both tissue factor expression and PI3K-Akt signaling, administration of inhibitors of PI3K-Akt signaling further enhances VEGF-induced tissue factor expression.131,132 Taken together, these data suggest that statins may inhibit blood coagulation, at least in part, through a selective activation of PI3K-Akt signaling in endothelial cells, leading to an inhibition of tissue factor expression.
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Integrated Regulation of Growth and Angiogenesis by Akt
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In addition to its role in angiogenesis, Akt has also been implicated
as a general regulator of tissue and organ growth. Studies in
Drosophila have demonstrated that components of the insulin/IGF
signaling pathway including Akt are involved in the regulation
of organ growth and body size in response to nutritional input.
134,135 The role of this pathway in vertebrate growth control is indicated
by the growth retardation observed after targeted disruption
of various components of this pathway in mice.
28,136,137 Importantly,
it has recently been shown that the targeted disruption of Akt1
gene in mice results in general growth retardation.
28,137
In higher animals, organ growth is accompanied by the recruitment of new blood vessels. The dual role of Akt signaling in angiogenesis and tissue growth suggests that both processes can be coordinately regulated by this signaling step during organ enlargement. Consistent with this notion, it has been demonstrated that exercise training, a well-known stimulator of muscle hypertrophy, enhances VEGF expression and increases vessel density in skeletal muscles, and that exercise-induced increase in vessel density is blocked by a VEGF-neutralizing antibody.138 Likewise, cardiac muscle cell-specific deletion of VEGF gene results in thin ventricular wall with fewer coronary vessels.139 These results indicate that angiogenesis associated with physiological muscle tissue growth is dependent on paracrine VEGF secretion. Based on the notion that Akt positively regulates organ growth, we have examined the hypothesis that Akt may be involved in VEGF secretion associated with muscle hypertrophy. Indeed, overexpression of Akt in skeletal muscles in vivo induces skeletal muscle hypertrophy, local VEGF production, and angiogenesis.140 Collectively, these findings suggest that Akt signaling in both muscle cells and endothelial cells coordinately regulate overall growth of muscle tissues in vertebrates. This concept may also be applicable to other organs as well.
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Conclusions
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PI3K-Akt signaling axis is activated by a variety of stimuli
in endothelial cells and regulates multiple critical steps in
angiogenesis, including endothelial cell survival, migration,
and capillary-like structure formation. Furthermore, this signaling
pathway also regulates cardiovascular homeostasis and vessel
integrity at least in part by controlling NO synthesis (
Figure 4).
Angiogenesis has been implicated in the pathophysiology
of a number of diseases, and regulation of angiogenesis, both
its increase and decrease, could be an important therapeutic
strategy for those disease states. Thus, further dissection
of the PI3K-Akt pathway and elucidation of the downstream effector
molecules will lead to a better understanding of blood vessel
growth and may provide avenues for the development of novel
therapeutic interventions.
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Figure 4. Activation of PI3K-Akt signaling axis in endothelial cells. Various growth factors, mechanical stimuli, and pharmacological interventions activate Akt signaling in endothelial cells, and Akt-dependent pathways control several events critical for cardiovascular homeostasis and angiogenesis.
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Acknowledgments
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This work was supported in part by NIH grants AR-40197, AG-17241,
HD-23681, AG-15052, and HL-50692. I.S. is supported by the Tanabe
Medical Frontier Conference and American Heart Association New
England Affiliate Fellowship Award.
Received March 27, 2002;
revision received April 30, 2002;
accepted May 6, 2002.
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Abstract
Introduction