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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Cycle. Author manuscript; available in PMC Sep 18, 2006.
Published in final edited form as:
Published online Mar 1, 2006.
PMCID: PMC1569947
NIHMSID: NIHMS9155

Akt1 in Endothelial Cell and Angiogenesis

Abstract

Akt, also known as Protein kinase B (PKB), regulates essential cellular functions such as migration, proliferation, differentiation, apoptosis, and metabolism. Akt influences the expression and/or activity of various pro- and anti-angiogenic factors and Akt isoforms (Akt1, Akt2 and Akt3) have been proposed as therapeutic targets for angiogenesis-related anomalies such as cancer and ischemic injury.

Keywords: Akt signaling, angiogenesis, endothelial cell, thrombospondin, collagen, laminin, vascular permeability

INTRODUCTION

Neovascularization, the process of building of new capillaries, is essential for physiological processes including embryonic development and tissue remodeling, but is also involved in pathological conditions such as tumor development.1,2 Neovascularization is triggered by pro-angiogenic factors like vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). These growth factors induce activation of various cell types through interaction with tyrosine kinase receptors. While endothelial cell (EC) migration and proliferation might be called “major angiogenic responses”, angiogenesis is a truly systemic process. It involves vascular leakage, hemostasis in tissues, activation of elements of immune system, recruitment of bone-marrow-derived cells, activation of proteases, and recruitment and proliferation of perivascular cells during vascular maturation. In ECs, the majority of growth factor-induced responses are mediated by the activation of the PI3 kinase-Akt signaling cascade.3 Downstream targets of this pathway and their specific roles in regulating cellular processes have been reviewed previously.4,3 Multiple biological activities are assigned to the VEGF-PI3Kinase-Akt signaling cascade in cell types involved in angiogenic response. Akt is thought to regulate cell survival5,6,7,8 and cell migration.3,5,9 In addition, Akt is believed to play a crucial role in glucose metabolism, cell cycle and transcriptional regulation, apoptosis, and tumor progression.10 The purpose of this article is to present the complexity of the Akt signaling network in ECs and discuss its role in angiogenesis.

ISOFORMS OF Akt

Mammalian cells are characterized by the expression of three different Akt isoforms (Akt1, 2, and 3; also known as PKBα, β, and γ), encoded by distinct genes.11 From gene knockout studies in mice, it is known that the loss of the Akt1 gene leads to organism growth retardation, accompanied by the reduced size of multiple organs, and elevated level of apoptosis in restricted cell types12,13 as well as impaired extra-embryonic vascular patterning and placental hypotrophy, and a partially penetrant phenotype of a higher fetal mortality.14 Akt2 gene disruption results in mild growth deficiency and impaired insulin signaling in the liver and skeletal muscle that leads to insulin resistance and diabetes.13,15 Akt3 knockout mice exhibit a predominantly neurological phenotype and have the reduced brain size.16 It is notable that Akt isoforms have a considerable functional overlap, as the deletion of both Akt1 and Akt2 genes results in severe growth retardation and multiple developmental defects leading to perinatal lethality.17 However, the differences in functional abnormalities observed in Akt1, Akt2 and Akt3 null mice suggest that the isoforms have physiologically diverse roles and/or are expressed differently in distinct cell types. More conclusive evidence regarding the role of Akt1 in vascular biology has been obtained from recent studies using animals that either lack or overexpress Akt15,18-22 (summarized in Table 1).

Table 1
Summary of effects of Akt1 manipulation in mouse models

Akt SIGNALING IN ENDOTHELIAL CELLS

Akt is expressed in different cell types and plays a central role in a variety of processes. The cellular responses to Akt phosphorylation are executed via signal transduction pathways initiated by phosphorylation of various Akt substrates. Akt has been implicated as an anti-apoptotic mediator in numerous cell death paradigms, including withdrawal of extracellular matrix, oxidative and osmotic stress, ischemic shock, irradiation and treatment of cells with chemotherapeutic agents.23-25 Additionally, specific roles have been assigned to Akt signaling in processes such as cell migration, and tumor progression and metastasis in multiple cell types.3,26 Among all isoforms, Akt1 seems to be predominantly expressed in ECs.5

Target substrates of Akt1 in endothelial cells

Since Akt phosphorylates numerous substrates in mammalian cells, the search for new Akt substrates in various cell types is a topic of intensive investigation. An array of Akt targets have been identified and linked to physiological functions of ECs (Fig. 1). Upon stimulation by growth factors, Akt activation occurs almost immediately and it subsequently regulates cellular processes involved in EC survival. Akt targets include Bad,27 Forkhead (FKHR) factors,28,29 IKKα,30 Mdm2 (in p53-mediated apoptosis),31 Yes-associated protein (YAP; in p73-mediated apoptosis),32 caspase9,33 and GSK3.34,35 In addition to its role in cell survival, GSK-3 appears to regulate the cell cycle,36 the trafficking and recycling of αvβ3 and α5β1 integrins37 and cell proliferation.3

Figure 1
Phosphorylation of Akt affects multiple substrates. Activation of Akt (phosphorylation on S473 and T308) leads to phosphorylation and activation/inactivation (shown by signs of inhibition and coactivation, respectively) of multiple substrates. Changes ...

It is important to take into consideration that Akt is also capable of controlling long-term cellular responses by regulating gene expression at the level of transcription and translation.4 In ECs, Akt regulates transcription of various genes through direct or indirect phosphorylation of various transcription factors4 and affects protein expression at the level of translation through the tuberous sclerosis complex (TSC/mTOR/4E-BP1/eIF4E).38 The TSC1/2 complex is multifunctional and was shown to affect EC proliferation through the TSC/mTOR/p70S6Kinase pathway.39 In addition, Akt regulates the activity of endothelial nitric oxide synthase (eNOS) via phosphorylation at Ser1177, regulating NO production and vasodilation.40

Another important function of Akt in ECs is its ability to modulate migration toward various matrix proteins in response to growth factors. One of the downstream targets of Akt in cell migration appears to be the Rho family of G proteins.41 Recently, p21 activated kinase (PAK), known to be responsible for myosin light chain phosphorylation and involved in cell migration, was identified as a substrate of Akt.42 Akt seems to be important for regulation of cell migration via actin reorganization,9 and through phosphorylation of a cytoskeletal protein called girdin.43 Integrin β3, an integrin present in ECs, is directly phosphorylated by Akt at Thr 75344 and this phosphorylation regulates binding of its adaptor protein Shc.45 Taken together, these reports establish the versatile nature of Akt in the regulation of EC function. However, due to the highly branched signaling network downstream of Akt, and different and sometimes opposite effects of short-term versus long-term Akt activation, its role in pathophysiological processes remains elusive. To complete an understanding of how various effects of Akt are reconciled in the regulation of EC functions, further studies of the roles of individual Akt isoforms and their downstream pathways are required.

Akt1 regulates endothelial cell functions in vitro

Our recent study5 using ECs isolated from Akt1−/− mice showed that Akt1 accounts for approximately 75% of the total Akt activity. Deficiency in Akt1 impaired the ability of ECs to form sprouts from aortic rings. In addition, Akt1−/− ECs exhibited impaired migratory responses toward matrix proteins such as fibronectin, fibrinogen, and vitronectin. Akt1−/− ECs exhibited a decreased proliferative response to serum compared to WT ECs. Another recent study20 also indicates the importance of Akt1 in migration and proliferation of ECs. We previously showed that VEGF induced activation of integrins (major mediators of cell migration), as quantified by the ability of ECs to bind soluble ligand fibrinogen.46 In a ligand-binding assay, Akt1−/−ECs bound 3-fold less fibrinogen than WT counterparts, thus illustrating impaired integrin activation in Akt1−/− ECs. This finding suggests that Akt1 is necessary for inside-out integrin signaling. Impaired integrin function often results in decreased extracellular matrix assembly and deposition;47 this, in turn, might affect the integrity of the endothelial monolayer and its permeability, an essential part of the angiogenic response.48 Our study5 showed that Akt1−/−EC monolayers were 50% more permeable compared to WT. Moreover, reduced integrin activation might be responsible for the altered composition of extracellular matrix in tissues and vessel walls observed in vivo models.5

Akt1 in the regulation of angiogenesis in vivo

It has been estimated that Akt kinase has over 9000 possible substrates.49 Therefore, Akt might regulate angiogenic responses by several distinct, and possibly antagonistic, mechanisms. VEGF is a major angiogenic factor secreted by cells and its crosstalk with Akt is very complex. First, Akt is known to mediate hypoxia-induced expression of VEGF in vitro and in vivo.50,51 Its role appears to be complex, however, since long-term activation of Akt causes a paradoxical decrease in VEGF expression and a decrease in the vascularity of cardiac tissues.19 Second, Akt is activated in ECs in response to exogenous VEGF.9 Finally, in addition to VEGF,52 Akt might affect the protein levels and activities of several key regulators of angiogenesis, including Angiopoetin 2.3 Recent studies clearly demonstrate that, due to the diversity of Akt downstream targets (Fig. 1), the Akt pathway requires precise and timely regulation.18,19

Two general approaches are available to study protein function in vivo: first, overexpression using a transgene (transgenic model); and second, abrogation, downregulation, or functional inhibition of the endogenous gene (knockout model transgenic animals that over-express a dominant-negative form of the molecule). These approaches have all been utilized to address the role of Akt in vivo (Table 1).

Akt can be present in cells in inactive and active (phosphorylated at T308 and S473) states. For this reason, one of the preferred transgenic models is the one that involves overexpression of the constitutively active, membrane-bound form of Akt (myrAkt).5,53 Expression of the transgene in certain cell types may be achieved by the placing it under the control of a cell-specific promoter.18,19,21 Regulation of the transgene expression by induction or repression of the promoter (e.g., a tetracycline-regulated system) has been used for Akt1 studies; this allows myrAkt1 expression to be regulated by feeding the animal a diet supplemented with tetracycline.19,18 These mouse models have certain advantages over other models due to the minimization of developmental compensatory effects. However, overexpression of a transgene often exceeds the naturally occurring levels of its active form: In a recent study, tissue-specific activity of myrAkt1 was increased 15-fold relative to Akt1 activity in control (WT) animals.19

At least two recent studies revealed that it is important to consider the acute/short-term and chronic/long-term effects of myrAkt1 expression19,21 (see Table 1). As an example, whereas short-term Akt activation in the heart resulted in increased angiogenesis, chronic activation led to decreased angiogenesis and increased fibrosis.19,21,54 A similar phenomenon was observed in Akt1 knockout mice: Short-term Akt1 deficiency affected the activation of eNOS in response to growth factors,5,20 whereas the long-term repression of Akt1-mediated signaling influenced the expression of extracellular matrix proteins in skin and blood vessels.5 These effects can be explained by the change in repertoire of downstream signaling molecules, leading to prevalence of pro- or anti-angiogenic signaling events (Fig. 2).

Figure 2
Akt1 signaling has both pro- and anti-angiogenic effects. The balance between signaling pathways under different conditions determines the angiogenic phenotype.

The knockout studies substantially supplemented the conclusions from reports based on overexpression with regard to role of Akt in vivo. The advantage of the knockout studies is that the loss-of-expression phenotype can be assessed. However, the interpretation of the observed phenotype might be complicated by compensatory changes during development. By definition, the knockout is a long-term absence of Akt activity; therefore, the phenotype of Akt null mice should be the opposite of the phenotype resulting from the long-term Akt activation. Indeed, we observed increased angiogenesis in Akt1−/− mice compared to WT mice in our recent study,5 while the decreased angiogenesis was observed upon prolonged Akt activation.19,21 Three different models of angiogenesis in vivo, the matrigel plug assay, a tumor angiogenesis assay, and adenoviral expression of VEGF in the skin, showed that pathological angiogenesis in Akt1−/− mice is enhanced compared to WT mice.5 We suggested that several mechanisms might be responsible for the phenomenon of increased angiogenesis in the Akt1−/− mice: changes in vascular permeability; a thinner blood vessel basement membrane with reduced laminin content; reduced expression of anti-angiogenic regulators, thrombospondins 1 and 2; and alterations in extracellular matrix density. Moreover, as discussed below, most of these features are linked by causal relationships.

Akt1-dependent pathways involved in the regulation of angiogenesis

Our studies suggest that Akt1-dependent regulation of the extracellular matrix environment might be a major reason for the differences between in vitro and in vivo experiments probing the role of Akt1 in angiogenesis. Extracellular matrix provides structural support to cells and also modulates their functions. In vitro assays, cells can migrate without any restraints. However, migration of endothelial and other cells in tissues requires softening and degradation of the matrix mesh by proteases (i.e., matrix metalloproteases or MMPs). Most of the studies of the role of Akt in cell migration have been performed in two-dimensional (2D) cell culture models in vitro. As the physical and biochemical characteristics of extracellular matrix play a significant role in the regulation of cellular functions, numerous discrepancies between cellular behavior in vitro and in vivo (3D) models were observed. It is known that in 2D in vitro cultures, cells form adhesion complexes at the leading edge and extend themselves forward via formation of lamellipodia or filopodia. However, it remains unclear whether a similar mechanism is operational in the 3D in vivo environment.55,56 Moreover, it is almost impossible to reconstitute a complexity of in vivo matrix in cell culture conditions.

In our recent publication, we assessed the levels of matricellular proteins thrombospondins (TSP1 and 2), collagen, and laminin in Akt1−/− mice and found them to be reduced.5 Thrombospondins might directly regulate angiogenesis via induction of EC apoptosis,57 control of vascular permeability5,58 or regulation of collagen matrix.59 In addition, it is known that the absence of TSP-2 expression results in impaired organization of collagen fibrils in the skin wounds60 and matrix abnormalities in the skin due to increased MMP-2 activity.61 TSP-1 has been shown to inhibit tumor growth by blocking the activity of MMP-9.62 Thus, collagen matrix abnormalities and the softer skin of Akt1−/− mice compared to WT mice might be a result of increased MMP activity, which in turn was caused by reduction in TSP-1 and TSP-2 expression.

Tumors grown in WT mice are more densely packed with a higher number of tumor cells per unit area compared to Akt1−/− mice. While reduced expression of TSPs in the skin, as well as reduced expression of laminin in the basement membrane, makes the Akt1−/− blood vessels leaky, disorganized collagen matrix is easier target for MMPs.5 Previous studies showed that collagen density is essential to restrain the growth of tumors.63,64 There is evidence that a cross-linked collagen prevents protease-dependent migration,65 whereas a noncross-linked collagen does not.66 Taken together, these findings emphasize the role of Akt in extracellular matrix assembly, which, in turn, regulates tumor growth and angiogenesis. Importantly, in double Akt1/Akt2 null mice, skin development was even more severely impaired than in Akt1−/− mice. A deficiency of two Akt isoforms leads to a translucent skin phenotype and the individual skin layers are much thinner than in WT mice.17 Thus, enhanced vascular permeability combined with the defective organization of extracellular matrix leads to enhanced angiogenesis in Akt1−/− mice.

In addition to factors discussed above, endothelial nitric oxide synthase (eNOS), a downstream target of Akt in ECs has been shown to be important for adaptive angiogenesis following hind limb ischemia in Akt1−/− mice.20 Although a reduced basal level of active eNOS is observed in Akt1−/− ECs, compensatory mechanisms via kinases other than Akt1, such as Protein kinase-A,67 are evident since stimulation with VEGF results in a partial recovery.5 As eNOS has been shown to be necessary for blood vessel maturation,68 the reason for smaller and immature blood vessels observed in tumors grown in Akt1−/− mice might be deficiency in levels of active eNOS. Interestingly, in the absence of VEGF stimulation Akt1−/− skin showed no differences from WT skin in blood vessel number, vessel area, or maturation, despite the observed decrease in basal levels of Akt activity.5 While reduction in nitric oxide levels is a short-term response, a long-term Akt1 deficiency results in thinner basement membrane of the Akt1−/− blood vessels compared to WT associated with decreased content of laminin.5 Another recent report also suggests that Akt can directly regulate the expression of laminin by mesangial cells in response to insulin-like growth factor 1.69 It was recently shown that deficiency of Akt1, but not Akt2, impairs revascularization and repair of the tissues followed by hind limb ischemia.20 VEGF production stimulated by hypoxia in this model is anticipated to be defective in Akt1−/− mice, since Akt is directly involved in the regulation of VEGF expression.52 This particular feature is distinct from other in vivo models of pathological angiogenesis, where VEGF is constantly supplied by implanted tumors, matrigel or delivered by VEGF-encoding adenovirus.5 Thus, while comparing results of different studies on angiogenesis one should take into consideration the differences between experimental models as well as endpoints of the studies. For example, our recent study shows that results obtained with angiogenic growth factors that vary in their potential to stimulate vascular leakage can be quite different.5

CONCLUSIONS AND PERSPECTIVES

Akt1 is vital for the regulation of EC functions, and maintenance of vascular integrity. Our results5 and other recent reports18,19,21 suggest that the level of active Akt1, as well as its short-term and long-term activation states, in vascular cells can regulate various signaling pathways to affect the balance of pro- and anti-angiogenic factors (Fig. 2). In our Akt1 knockout model system,5 changes in the expression and organization of extracellular matrix proteins, such as collagen and laminin, reduced expression of TSP-1 and TSP-2, increased vascular permeability, and decreased vessel maturation conferred an enhanced angiogenic phenotype upon VEGF stimulation.

Akt kinase has the potential to phosphorylate over 9000 proteins;49 the Akt-driven signaling network is capable of regulating cellular processes by modulating transcription and translation, and by modifying proteins at the posttranslational level. Due to the complexity of Akt involvement in various cellular processes, the downstream effectors of Akt that are the most critical to the pro- or anti-angiogenic roles of Akt remain to be determined.

The most recent studies raise the question about the isoform-specific properties of Akt in the context of different cell types. Akt1 was shown to negatively regulate migration of breast cancer cells through transcription factor NFAT.70,71 In a model using epithelial cells, Akt1 negatively regulated migration and downregulation of Akt2 decreased proliferation and cell survival.72 Hence, the functions of individual isoforms of Akt must be considered when designing therapeutic drugs involving Akt.

In light of the observed time-dependent switch of Akt1 signaling from pro-angiogenic to anti-angiogenic in the vasculature and heart,19 it is important to identify what downstream effector molecules are responsible for these effects. This information will provide us with new and more specific molecular targets for pro- or anti-angiogenic therapy. The in vivo studies listed in Table 1 have important implications for development of therapeutic strategies to minimize the consequences of heart failure and myocardial or other organ ischemia and for implementation of effective anticancer therapy regimens via manipulation of Akt activity. It is critical that specific pathways or substrates of Akt are targeted, since inhibition of Akt itself affects a wide range of intracellular processes. Also, it is clear that our understanding of cell-type specific and time-dependent Akt signaling is far from complete. Although it appears that Akt is a very attractive therapeutic target for various aspects of vascular abnormalities, the complexity and somewhat antagonistic effects of Akt signaling should be taken into careful consideration.

Recently, the role of Akt kinase in cardiovascular biology has become a topic of intensive investigation. It is increasingly evident that Akt is central to regulation of multiple pathways in ECs, fibroblasts, and cardiomyocytes and therefore, its activity should be monitored/manipulated in course of treatment of ischemic conditions associated with myocardial infarction and stroke.

ACKNOWLEDGEMENTS

This work was supported by grant number HL071625 to T.V.B.

ABBREVIATIONS

VEGF
Vascular endothelial growth factor
FGF
Fibroblast growth factor
EC
Endothelial cell
TSP
Thrombospondins
PKB
Protein kinase B
WT
wild-type
MMP
matrix metalloproteases
2D, 3D
two- and three-dimensional, respectively
ENOS
endothelial nitric oxide synthase
IRI
ischemia-reperfusion injury
DN
dominant-negative

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