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The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis.

PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. We used the Cre-loxP system to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePten(flox/+) mice displayed enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect was partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePten(flox/+) endothelial cells showed enhanced proliferation/migration. Tie2CrePten(flox/flox) mice died before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and were associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis.

Normal cardiovascular development requires communication between endothelial cells and surrounding mesenchymal cells (Suri et al. 1996;Hellstrom et al. 2001). The vascular wall is composed of endothelial cells that surround the lumen of the vessel, backed by layers of pericytes (PCs) in microvessels, or vascular smooth muscle cells (vSMCs) in large vessels (Hungerford and Little 1999). Cross-talk between endothelial cells and PCs or vSMCs is critical for vascular remodeling and maturation. Defects in PC/vSMC recruitment, such as result from mutations abrogating signaling via PDGF-B (Lindahl et al. 1997), Ang-1 (Suri et al. 1996), or TGF-␤ (Dickson et al. 1995), result in vascular abnormalities.
PTEN is important for normal cardiovascular homeostasis. In vitro, PTEN inhibits vascular sprouting and endothelial tube formation induced by VEGF; dominantnegative mutation of PTEN abolishes these effects (Huang and Kontos 2002). Su et al. (2003) have shown that tumor angiogenesis and tumor growth in vivo are blocked by Pten overexpression in tumor cells or by administration of PI3K inhibitors. However, it was not clear in that study whether the inhibition of tumor growth was caused by the angiogenesis defect or by a direct effect on the tumor cells. Here we demonstrate that the role of PTEN in cardiovasculogenesis and angiogenesis is to regulate the expression of vascular signaling molecules, particularly VGFs.

Generation of endothelial cell-specific Pten-deficient mice
We generated a conditional Pten knockout mouse in which Pten expression was governed by Tie2, an endothelial cell-specific promoter. Pten exon 5, which encodes the phosphatase domain, was flanked with loxP sequences (Ptenflox) ( Fig. 1A; Suzuki et al. 2001). Pten flox/flox males were crossed with Pten flox/+ females that carried a single Tie2Cre transgenic locus (Koni et al. 2001) coupled to a reporter transgene (CAG-loxP-CAT-loxP-EGFP) (Kawamoto et al. 2000). Analysis of reporterpositive Tie2Cre progeny showed that recombination occurred mainly in endocardial cells and systemic endothelial cells but not in PCs/vSMCs (Fig. 1B). PCR examination of DNA from embryonic day 9.5 (E9.5) cells positive for the endothelial cell marker VEGFR2 confirmed that efficient Cre-mediated recombination had occurred in the Tie2CrePten flox/flox mice. Quantitation of recombination was established in preliminary PCR experiments using mixtures of various ratios of Pten ⌬ and Pten flox plasmid DNAs under identical PCR conditions (Fig. 1C). The recombination frequency in VEGFR2 + cells of Tie2CrePten flox/flox mice was ∼95% (Fig. 1D).

Increased angiogenesis and accelerated tumor growth in Tie2CrePten flox/+ mice
Histological analyses of systemic vessels and the heart revealed no significant structural differences between Tie2CrePten +/+ and Tie2CrePten flox/+ mice (data not shown). Since VGFs activate the PI3K-PKB/Akt pathway, we investigated whether VGF-stimulated angiogenesis was increased in Tie2CrePten flox/+ mice. Matrigel implants impregnated with bFGF, bFGF + VEGF, bFGF + Ang-1, or PBS were administered subcutaneously to Tie2CrePten +/+ and Tie2CrePten flox/+ mice. Blood vessel infiltration of the implants was quantified by immunostaining with Figure 1. Generation of endothelial cell-specific Pten-deficient (Tie2CrePten flox/flox ) mice. (A) Targeting strategy. Exons of the murine Pten gene are represented by Ⅲ, and loxP sites are indicated by black arrowheads. The floxed (Pten flox ) and deleted (Pten ⌬ ) alleles are shown. Primers used for genotyping are shown as red arrows (a-c). (B) Tissue distribution of Tie2Cre-mediated recombination. Immunohistochemistry was used to evaluate GFP expression at E9.5 (panels a,b), and GFP plus SMA (red) expression in the dorsal aorta (panel c) and heart (panel d) at E10.5, in Tie2Cre Tg mice carrying the reporter transgene. (C) Quantitation of genomic PCR. A total of 1 fg of a mixture of various ratios of Pten ⌬ and Pten flox plasmid DNAs was used as template DNA. (D) Deletion of the Pten gene. Genomic PCR of DNA from VEGFR2 + cells in E9.5 embryos of the indicated genotype. In Tie2CrePten flox/flox mice, the vast majority of VEGFR2 + cells showed deletion of the Pten gene. PCR conditions were identical to those in C.
We next used PCNA staining and the Transwell system to evaluate the proliferation and migration, respectively, of mouse lung endothelial cells (MLECs) from 10wk-old Tie2CrePten +/+ and Tie2CrePten flox/+ mice. In response to stimulation with Ang-1 or VEGF-A, total numbers of PCNA + cells and their migration were significantly increased in Tie2CrePten flox/+ cultures compared with controls (Fig. 2F).
Because Tie2 is expressed in hemangioblasts as well as in endothelial cells, Tie2Cre also deletes the Pten gene in hematopoietic cells. However, numbers and subsets of T cells, NK cells, and NKT cells were all normal in Tie2CrePten flox/+ mice (Supplementary Fig. 2A,B). Tie2CrePten flox/+ splenocytes stimulated with either IL-12 or anti-CD3 plus anti-CD28 showed normal IFN-␥ secretion ( Supplementary Fig. 2C). Because macrophages are crucial for pathological angiogenesis (Bingle et al. 2002), we repeated the above Matrigel and tumor cell implantation experiments using LysMCrePten flox/+ mice. In these mutant animals, the Pten gene is heterozygously deleted in >80% of peritoneal macrophages (data not shown). No differences in tumor angiogenesis were observed between the LysMCrePten +/+ and LysMCrePten flox/+ genotypes (Supplementary Fig. 2D-F). Thus, the accelerated tumor growth in Tie2CrePten flox/+ mice results from enhanced angiogenesis driven by increased endothelial cell proliferation/migration and not from defects in macrophages or tumor immunosurveillance. The life expectancy, fertility, and gross appearance of viable F1 and F2 Tie2CrePten flox/+ mice appeared normal, but no Tie2CrePten flox/flox mice were found among 108 offspring from Tie2CrePten flox/+ and Pten flox/flox intercrosses. Genotyping of 230 progeny of these intercrosses at various embryonic stages showed that homozygous mutant embryos were present at the expected Mendelian frequency up to E9.5, but that resorption commenced at E10.5 and embryonic loss occurred at E11.5 (Table 1).
Histologically, E8.25 Tie2CrePten flox/flox embryos were essentially normal with respect to gross appearance of the central vascular tree (Fig. 3A, panels a,b), including the rostral-caudal aorta (red arrows), and the anterior and posterior cardiac veins (Fig. 3A, panels c-f, blue arrows). Pten is thus dispensable for the differentiation of angioblasts from the ventral mesoderm, their appropriate migration within the embryo, and their alignment to form major vessels. However, most E8.25 homozygous mutants exhibited delayed heart looping ( Fig. 3A, panels g,h, yellow arrows) and enlarged and partially fused yolk sac vessels (Fig. 3A, panels i,j; green arrow). By E9.0, the mutant allantois had connected normally to the chorion (Fig. 3B), but by E9.5, the capillary plexus was enlarged (Fig. 3C, panels a,b, yellow arrows) and distinct branches of large vessels such as the anterior cardinal vein were not formed (Fig. 3C, panels c,d, red arrow) because of a failure in primary vascular plexus remodeling. Vascular sprouting into the neural tube was barely detectable (Fig.  3C, panels e,f, blue arrow). Tie2CrePten flox/flox embryos also failed to generate distinct vitelline vessels in the yolk sac (Fig. 3D, panels a-d), showing instead a meshwork of interconnected, oversized endothelial cell-lined tubes (Fig. 3D, panels e,f). Dilated capillary-plexus vessels filled the intercapillary spaces ( Fig. 3D, panels g,h).
Growth retardation was observed in 50% of E9.5 Tie2CrePten flox/flox embryos. By E10.5, the majority of mutants showed profound growth retardation as well as pericardial cavity enlargement and frequent bleeding into the pericardial cavity (Fig. 3E, panels a-f, red arrows) or large trunk vessels (Fig. 3E, panel g, blue arrows). Numbers of primitive hematopoietic cells in Tie2CrePten flox/flox blood vessels were normal from E8.25 to E10.5 (data not shown).
Morphometric analyses showed that vessels in Tie2CrePten flox/flox yolk sacs were fewer in number (Supplementary Fig. 3A) but larger in diameter (Supplementary Fig. 3B) than in controls. Tie2CrePten flox/flox yolk sacs also showed an increase in endothelial cell numbers ( Supplementary Fig. 3C) that was attributable to proliferation, as judged by their increased staining for Ki67, a nuclear protein highly expressed in proliferating cells ( Supplementary Fig. 3D). TUNEL assays revealed no significant differences in numbers of apoptotic endothelial cells in mutant and wild-type yolk sacs (data not shown). No vascular abnormalities were evident in E8.5-E11.5 Tie2CrePten flox/+ embryos. Thus, enhanced endothelial cell proliferation and impaired vascular remodeling are likely responsible for the lethal vascular phenotype of Tie2CrePten flox/flox embryos.

Impaired recruitment of PCs and vSMCs and defective cardiac muscle development in Tie2CrePten flox/flox mice
We examined PC and vSMC recruitment using immunostaining with anti-smooth muscle antigen antibody (␣SMA). At E10.0, ␣SMA + cells were observed in perivascular regions of the Tie2CrePten +/+ yolk sac (Fig. 3F, panel a) and in the placental extraembryonic circulation (data not shown). In contrast, no ␣SMA + PCs/vSMCs were observed adjacent to endothelial cells in Tie2CrePten flox/flox vessels either in the yolk sac ( Fig. 3F, panel b) or placenta (data not shown). In the embryo proper, ␣SMA staining observed in the vessels, heart, and dorsal aorta of wildtype embryos (Fig. 3F, panels c,e) was dramatically reduced in Tie2CrePten flox/flox embryos (Fig. 3F, panels d,f). Electron micrographs of E9.5 Tie2CrePten flox/flox yolk sacs and placenta confirmed the absence of PCs around the capillary network endothelium (Fig. 3F, panel h). The observed bleeding from the large trunk vessels may result from the impaired recruitment of PCs/ vSMCs to these tissues. A reduction in the size of the developing intraventricular septum and cardiac trabeculae, and a thinning of the cardiac wall (Fig. 3F, panel f), were also observed in homozygous mutants. The thinnest cardiac walls resembled membranes (Fig. 3F, panel f, blue arrow) such that bleeding in the mutant pericardial cavity could have resulted from cardiac wall rupture or leakage of blood cells from the cardiac lumen. Because Genotypes of mice at indicated post-natal (P) or embryonic (E) days were determined using PCR analysis. Numbers in parentheses indicate the total number of abnormal Tie2CrePten flox/flox embryos counted under a microscope. a Embryos could not be genotyped because of resorption.

GENES & DEVELOPMENT 2057
Cold Spring Harbor Laboratory Press on July 22, 2018 -Published by genesdev.cshlp.org Downloaded from the Tie2CrePten flox/flox vascular phenotype precedes the development of a functional circulation and the onset of growth retardation, these defects are most likely due to the Pten mutation and are not secondary to circulatory failure, ischemia, or growth retardation.
In Tie2CrePten +/+ MLECs, expression levels of VEGF-A and its receptors VEGFR1 and VEGFR2 were significantly increased after stimulation with VEGF-A, as expected. However, this expression was further increased in Tie2CrePten flox/+ MLECs (Fig. 4F). This enhanced expression of VEGF-A and its receptors may thus partly contribute to the enhanced angiogenesis observed in Tie2CrePten flox/+ mice.
Homozygous mutation of p85␣ restored control of angiogenic responses in Tie2CrePten flox/+ mice to the same degree as mutation of p110␥ (Fig. 6A-D). However, the defect in cardiovascular morphogenesis seen in Tie2CrePten flox/flox mice was only partially rescued by p85␣ deficiency (Fig. 6E) such that these animals survived only until E14.5-E15.5.
Our data show that the enhanced angiogenesis exhibited by Tie2CrePten flox/+ mice involves both p85␣ and p110␥, while the defect in cardiovascular morphogenesis in Tie2CrePten flox/flox mice depends more strongly on p110␥ than on p85␣. We conclude that the interaction of PI3Ks and Pten is essential for the regulation of cardiovascular morphogenesis and post-natal neovascularization, including tumor angiogenesis.

Heterozygous Pten deficiency in endothelial cells accelerates tumor growth by enhancing tumor angiogenesis
Angiogenesis is a tightly regulated event critical for tumor growth. Tumor angiogenesis requires the activation, proliferation, and migration of endothelial cells, tube formation, and tissue infiltration by preexisting blood vessels. All these processes are controlled by angiogenic growth factors secreted either by the tumor or the surrounding stroma (Folkman and Shing 1992;Plate et al. 1994). Overexpression of VEGF, Ang-1, bFGF, and their receptors is strongly associated with invasion and metastasis in human cancers, and VGF inhibition blocks angiogenesis and tumor growth (Kim et al. 1993).
Our study is the first to demonstrate that the loss of Pten specifically in mouse endothelial cells makes them hypersensitive to VGFs and thus is responsible for enhanced angiogenesis leading accelerated tumor growth. An identical mechanism may be operating in humans with Cowden disease, a hereditary syndrome of cancer susceptibility caused by heterozygous mutations of PTEN. Our results have provided fresh insight into this syndrome and suggest that an individual who inherits a mutated PTEN allele is not only at risk for additional tumorigenic mutations due to the LOH of PTEN function but may also experience accelerated growth of any incipient tumors due to enhanced angiogenesis. Our findings thus further justify efforts to target PTEN and the PI3K-PKB/Akt pathway as a cancer therapy.

Homozygous Pten deficiency in endothelial cells causes a fatal vascular remodeling defect
The major downstream target of Pten is the survival kinase PKB/Akt, and PKB/Akt overexpression supports endothelial cell survival in vitro (Gerber et al. 1998). Transient activation of PI3K-PKB/Akt and subsequent acti-

GENES & DEVELOPMENT 2059
Cold Spring Harbor Laboratory Press on July 22, 2018 -Published by genesdev.cshlp.org Downloaded from vation of eNOS by growth factors such as VEGF enhance endothelial cell migration, angiogenesis, tube formation, and vasorelaxation (Shiojima and Walsh 2002). Constitutively active PKB/Akt (myrAkt) expression in vivo causes fatal vascular malformations and bleeding due to a failure in vascular remodeling (Sun et al. 2005). Mice deficient for Foxo1, a transcription factor negatively regulated by PKB/Akt, also show fatal vascular remodeling and cardiac defects (Furuyama et al. 2004). These findings are strikingly similar to those obtained for our endothelial cell-specific Pten-deficient mice. The phenotypes observed in Foxo1-deficient mice were attributed to reductions in connexin 37, connexin 40, and ephrinB2 expression (Furuyama et al. 2004). Indeed, we also found decreased connexin 40 and ephrinB2 expression in our mutants. Mice deficient for ephrinB2 die at E10.5 due to defects in vascular remodeling and sprouting that lead to pericardial effusion or bleeding (Wang et al. 1998). Taken together, these data suggest that dysregulation of downstream targets of PKB/Akt and Foxo1 due to loss of Pten function may be responsible for the lethal phenotype of Tie2CrePten flox/flox mice.
The recruitment of PCs/vSMCs may determine the transition from an immature to a mature capillary network (Jain 2003) via a process regulated by factors such as PDGF (Lindahl et al. 1997), Ang-1 (Suri et al. 1996), and TGF-␤1 (Dickson et al. 1995). If the gene dosage of PDGF-B is reduced by half, a modest increase in acellular capillary formation occurs (Lindblom et al. 2003). However, both PDGF-B and its receptor PDGFR␤ were normal in Tie2CrePten flox/flox mice. Disruption of Ang-1 or its receptor Tie2 in knockout mice causes vascular defects due to impaired recruitment of PCs/vSMCs (Dumont et al. 1994;Suri et al. 1996). The effects of Ang-1 are antagonized by Ang-2 since Ang-2 inhibits the autophosphorylation of Tie2 induced by Ang-1 (Maisonpierre et al. 1997). Indeed, administration of Ang-2 causes a dose-dependent PC "dropout" in the normal retina (Hammes et al. 2004). In our mutant mice, Ang-2 expression was markedly increased while that of Ang-1 was reduced, perhaps explaining the impaired recruitment of PCs/vSMCs. Normally, the interaction of endothelial cells and PCs/vSMCs drives expression of TGF-␤1 (Antonelli-Orlidge et al. 1989). Mice lacking either TGF- ␤1 or its receptor show abnormal capillary structure due to defective angiogenesis possibly associated with impaired PC/vSMC differentiation (Dickson et al. 1995;Larsson et al. 2001). However, TGF-␤1 was increased rather than decreased in our Tie2CrePten flox/flox mice. PKB/Akt activation inhibits Smad3, which eventually blocks TGF-␤ signaling (Remy et al. 2004). However, when we examined the expression of PAI-1, a downstream target molecule widely used to detect TGF-␤ activation, it was clear that such a mechanism was not operating in Pten-deficient endothelial cells at midgestation.
VEGF is a multifunctional VGF that stimulates the migration, proliferation, and survival of endothelial cells and promotes microvascular permeability and angiogenesis. VEGF also recruits PCs to the endothelial plexus (Benjamin et al. 1998). The expression of VEGF-A and its receptors was increased in Tie2CrePten flox/flox mice, consistent with a previous report in which activation of the PI3K pathway led to augmented VEGF-A expression (Jiang et al. 2000). Increased VEGF expression during lung organogenesis overstimulates endothelial cell growth and leads to abnormally large capillaries (Zeng et al. 1998). Mice overexpressing VEGF die at E14 due to cardiac abnormalities (Miquerol et al. 2000). We specu-late that the Pten deficiency in our homozygous mutant mice contributed to increased VEGF signaling, leading to large capillary lumens with increased numbers of endothelial cells as well as a thin myocardium in the heart.
VCAM-1 signaling is also important for cardiovascular development, and VCAM-1 deficiency causes pericardial bleeding and impaired cardiomyocyte development (Kwee et al. 1995). The dramatic reduction of VCAM-1 expression in Tie2CrePten flox/flox embryos might partially explain their cardiac phenotypes. Defects in additional VGFs, their receptors, or cell adhesion molecules acting downstream of PI3K and PKB/Akt may also contribute to the cardiovascular defect.
Pten is deleted only in the endocardium and not in the myocardium of Tie2CrePten flox/flox mice. However, Tie2CrePten flox/flox mice showed a thin myocardial layer, in contrast to the well-developed trabecular folds observed in myocardium-specific Pten-deficient mice (Crackower et al. 2002). We speculate that this discrepancy arises because the abnormal endocardium of Tie2CrePten flox/flox mice cannot provide the appropriate inductive interactions for proper development of the underlying myocardium (Suri et al. 1996). (E) Whole mounts of E10.5 and E14.5 embryos of the indicated genotypes showed that loss of p85␣ partially restored the development of Tie2CrePten flox/flox embryos. However, due to extensive bleeding, these embryos did not survive beyond E14.5-E15.5. For B and D, quantitative results are expressed as the mean ± SEM and are representative of four trials. Statistical differences were determined using the Student's t-test; (*) p < 0.05.

Mutation of p85␣ or p110␥ partially rescues endothelial cell-specific Pten defects
The precise functions of PI3K isoforms in endothelial cells have been difficult to ascertain. Because most VGF receptors have tyrosine kinase activity, class IA PI3Ks likely play major roles in cardiovasculogenesis and tumor angiogenesis. Indeed, endothelial cell growth/survival and angiogenesis are enhanced following ectopic expression of constitutively active p110␣, the catalytic subunit of class IA PI3Ks (Jiang et al. 2000). Consistent with this finding, the enhanced angiogenesis and accelerated tumor growth observed in our Tie2CrePten flox/+ mice, and the impaired cardiovascular morphogenesis observed in our Tie2CrePten flox/flox mice were partially resolved by loss of p85␣, the major regulatory subunit of class IA PI3Ks.
In this study, we shed light on the potential roles of the class IB PI3K in cardiovascular morphogenesis and post-natal angiogenesis. We generated double mutant mice lacking both Pten and class IB PI3K functions and demonstrated that the post-natal angiogenic responses of Tie2CrePten flox/+ mice were rescued to the same degree by loss of p110␥, the catalytic subunit of PI3K␥, as by loss of p85␣. Furthermore, compared with p85␣ deficiency, p110␥ deficiency dramatically resolved the defective cardiovasculogenesis observed in Tie2CrePten flox/flox mice. In the p85␣-deficient mice used in this study, only the p85␣ isoform was deleted, not its alternative splicing isoforms p55␣ and p50␣. Moreover, p85␤ and p55␥, the alternative regulatory subunits of class IA PI3Ks, still exist in these mice. However, since p85␣ is the major regulatory subunit of class IA PI3Ks, we believe that the class IB PI3K may have a more important function in cardiovasculogenesis than do the class IA PI3Ks.
We did not expect the enhanced angiogenesis induced in Tie2CrePten flox/+ mice by RTK agonists (e.g., VEGF and Ang-1) to be partially rescued by p110␥ deficiency. Up until now, p110␥ has been postulated to be activated downstream of GPCR but not downstream of RTK. Indeed, the activation of PKB/Akt and MAPK induced by VEGF and Ang-1 was not suppressed by p110␥ deficiency in vitro (Supplementary Fig. 4A). It is thus unlikely that an RTK type VGF receptor directly couples with p110␥, an interaction noted for PDGF receptors and erythropoietin receptors (Kaplan-Albuquerque et al. 2003). It may be that, in vivo, an unknown VGF (possibly a GPCR ligand) activates p110␥ and influences RTK signaling that is initiated by VEGF or Ang-1 and leads to angiogenesis. In endothelial cells, identified GPCR ligands include sphingosine-1-phosphate (S1P), angiotensin II, CXCL-16, and shear stress (Stoyanov et al. 1995;Dimmeler et al. 1998). One of the candidate GPCR ligands that may activate p110␥ in endothelial cells is S1P. S1P induces endothelial cell proliferation, migration, and morphogenesis in vitro and in vivo (Lee et al. 1999). Moreover, EDG1, a GPCR-type S1P receptor, is essential for vascular maturation (Liu et al. 2000). We have shown here that p110␥ deficiency partially blocks enhanced angiogenesis driven not only by Ang-1 or VEGF-A but also by S1P (Supplementary Fig. 4B). However, S1P-induced activation of PKB/Akt and MAPK was not suppressed by p110␥ deficiency (Supplementary Fig. 4A), indicating that the major downstream target of S1P may not be p110␥.
Although PTEN has dual lipid and protein phosphatase activities (Maehama and Dixon 1998;Li and Sun 1997), our results clearly demonstrate that a primary function of PTEN is to fine-tune the intracellular level of PIP3 produced by PI3Ks and thereby regulate vascular remodeling and tumor angiogenesis. Our data also suggest the functional overlapping of class IA and IB PI3Ks in angiogenesis. This hypothesis is supported by the lack of an endothelial cell phenotype in mice lacking p85␣ (Fruman et al. 1999;Terauchi et al. 1999), p85␤ (Ueki et al. 2002), p110␤ (Bi et al. 2002), or p110␥ (Sasaki et al. 2000) or in p110␦ "kinase dead" knock-in mice (Okkenhaug et al. 2002). Furthermore, our results demonstrate that, among the multiple PIP3 phosphatases, PTEN has an exclusive role in down-regulating PIP3 in endothelial cells. Various PI3K␥-specific inhibitors that are under investigation as anti-inflammatory drugs may therefore also be useful as cancer therapies targeting tumor angiogenesis.
Our study is the first report of the functional analysis of Pten and PI3K␥ in murine endothelial cells in vivo. We have demonstrated that the normal function of the PI3K-PKB/Akt-Pten pathway in endothelial cells is required for cardiovascular development, and that loss of Pten-mediated control of this pathway enhances tumor angiogenesis. Deficiency in Pten function thus contributes both to susceptibility to new tumorigenic mutations and to accelerated tumor growth. Inhibition of the PI3K pathway, including PI3K␥, is thus an attractive therapeutic target for the treatment of various malignancies.
Homologous recombination was detected using a GFP reporter gene as described previously (Kawamoto et al. 2000). Briefly, the reporter transgene contains the CAG promoter, the loxP-flanked CAT gene with an SV40 polyadenylation signal, and the GFP gene at the 3Ј end (STOP flox -GFP). Mice carrying both the Tie2Cre and STOP flox -GFP transgenes were generated to identify cells in which recombination had occurred. Flk-LacZ knock-in mutant mice have been described previously (Yamaguchi et al. 1993). Interbreeding of Tie2CrePten flox/+ and Flk-LacZ heterozygous mutant mice generated Pten homozygous and Flk-LacZ heterozygous double mutant mice. Littermate embryos were used for all experiments. The Institutional Review Board of the Akita University School of Medicine approved all animal experiments.

PCR analysis of Pten genotypes
Genomic DNA from mouse tails was isolated and amplified by PCR as described (Suzuki et al. 2001

RT-PCR
To assay VGF expression, RNA from 10 4 cells was extracted using an RNeasy Mini kit (Qiagen, Inc.) and reverse transcribed using an Advantage RT-for-PCR Kit (Clontech). Sequences of specific primers and amplified product sizes are listed in Supplementary Table 1.

Matrigel plug assays
Matrigel plug assays were performed as described previously (Prewett et al. 1999). Six-week-old mice were injected subcutaneously at the abdominal midline with 0.5 mL Matrigel (BD Biosciences) supplemented with 250 ng bFGF (R&D Systems) and/or 5 ng VEGF-A (VEGF165; PeproTech EC Ltd.) and/or 150 ng Ang-1 (provided by G.Y. Koh, KAIST, Deajeon, Korea). Control mice were injected with PBS-Matrigel. Mice were sacrificed 14 d after Matrigel injection, and vascularization was quantitated by immunohistological examination using anti-PECAM-1/CD31 antibody (Pharmingen) and a horseradish peroxidaseconjugated anti-rat Ig secondary antibody (Biosource International). Samples were then incubated with PBS containing 0.3 mg/mL diaminobenzidine (Dojin Laboratories) in the presence of 0.05% NiCl 2 for 10-30 min at room temperature, followed by the addition of 0.01% hydrogen peroxide. Infiltration of capillaries into the Matrigel was visualized by anti-CD31 staining and quantitated using NIH image software.

Cell proliferation and transmigration assays
Cell proliferation in response to 24 h stimulation with Ang-1 (300 ng/mL) or VEGF-A (10 ng/mL) was evaluated by PCNA immunostaining as previously described (Zhang et al. 2004). Cell transmigration assays were performed as described (Ito et al. 2003) using MLECs from 8-10-wk-old Tie2CrePten +/+ and Tie2CrePten flox/+ mice. Migration was measured 4 h after the addition of Ang-1 (300 ng/mL) or VEGF-A (10 ng/mL) to the lower chamber of a Transwell.

Whole-mount embryo immunostaining and LacZ staining
Immunostaining was conducted using anti-CD31 antibody or anti-smooth muscle actin antibody (1A4; Dako Cytomation). For LacZ staining, embryos and yolk sacs were fixed in cold 4% paraformaldehyde in PBS for 10 min, rinsed twice with PBS, and stained from 2 h to overnight at 37°C in X-Gal buffer (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl 2 , and 1 mg/mL X-Gal in PBS at pH 7.2).

Electron microscopy
The placentae and yolk sacs of E9.5 embryos were isolated, washed with washing buffer (3.5% sucrose in 0.1 M sodium cacodylate buffer at pH 7.3), post-fixed in buffered osmium tetroxide, and embedded in epoxy according to standard procedures. Ultrathin sections were stained in uranyl acetate and lead citrate and examined using a 100CX electron microscope operated at 60 kV (JEOL)