Generation of endothelial cell-specific Pten-deficient (Tie2CrePten^flox/flox) mice. (A) Targeting strategy. Exons of the murine Pten gene are represented by ▪, and loxP sites are indicated by black arrowheads. The floxed (Pten^flox) and deleted (Pten^Δ) 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^Δ 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.

Tie2CrePten^flox/+ mice show increased angiogenesis and accelerated tumor growth. (A,B) Increased angiogenesis in response to VGFs. (A, top) Representative photos of Matrigel plugs containing no growth factors (No), bFGF, bFGF + Ang-1, or bFGF + VEGF-A. Plugs were removed from Tie2CrePten^+/+ (+/+) and Tie2CrePten^flox/+ (flox/+) mice at 14 d post-injection. Plug vascularization is accelerated in the mutant. (Bottom) Anti-CD31 staining of sections of the Matrigel plugs in the top panel. Numbers of endothelial cells and vessel structures are increased in the mutant. (B) Quantitated vascularization of Matrigel plugs. The area of a plug staining positively with anti-CD31 was quantitated using NIH image software. Significant (*) increases in plug vascularization in response to VGFs were observed in Tie2CrePten^flox/+ mice (flox/+; n = 8) compared with wild-type littermates (n = 8). (C–E) Increased tumor growth. (C, top) Representative photos of LCC cells at 2 wk post-implantation in Tie2CrePten^+/+ and Tie2CrePten^flox/+ mice. (Bottom) Anti-CD31 staining of sections of the tumors in the top panels, showing an increased number of endothelial cells and vessel structures in the mutant. (D) Tumor volumes 1–3 wk after transplantation of B16BL6 melanomas (n = 4 mice/genotype) or LLC cells (n = 6 mice/genotype). (E) Quantitation of tumor vascularization 2 wk after implantation of tumor cells in D. (F) Enhanced proliferation and migration of Tie2CrePten^flox/+ MLECs after stimulation by Ang-1 or VEGF-A. (Top) Proliferation of VGF-stimulated MLECs as measured by PCNA immunostaining. (Bottom) Migration of MLECs cultured in Transwell cultures for 4 h in the presence of the indicated VGF. For B, D, E, and F, results are expressed as the mean ± SEM and are representative of three trials. Statistical differences were determined using the Student's t-test; (*) p < 0.05.

Cardiovascular defects in Tie2CrePten^flox/flox embryos. (A) Whole mounts of E8.25 embryos immunostained with anti-CD31. (Panels a–f) Vascular tree components, including the rostral-caudal aorta (red arrows) and anterior and posterior cardiac veins (blue arrows), were grossly normal in Tie2CrePten^flox/flox embryos. (Panels g–j) Delayed heart looping (yellow arrows) and enlarged and partially fused yolk sac vessels (green arrow) were apparent in the mutant. (B) Normal chorioallantoic fusion in E9.0 Tie2CrePten^flox/flox embryos. (C) Gross abnormalities in E9.5 Tie2CrePten^flox/flox embryos. Anti-CD31 staining of whole-mount (panels a,b), gross whole-mount (panels c,d), and histological LacZ staining (panels e,f) of E9.5 embryos are shown for Tie2CrePten^+/+ and Tie2CrePten^flox/flox embryos carrying the Flk-1^+/-/LacZ transgene. The mutant shows an enlarged capillary plexus (yellow arrows; panels a,b), a failure to remodel distinct branches of the anterior cardinal vein (red arrow; panel c), and impaired vascular sprouting into the neural tube (bottom, blue arrow; panel e). (D) Abnormalities in the E9.5 yolk sac. Whole mounts of E9.5 yolk sac (panels a,b), and anti-CD31 staining of yolk sac tissues (panels c–f), and H&E staining of yolk sac tissues (panels g,h) are shown. (Panels a–d) A failure of vascular remodeling to form mature distinct vitelline vessels can be clearly seen in the homozygous mutant yolk sac. (Panels e–h) Interconnected and homogeneously dilated endothelial cell-lined tubes are present. (E) Fatal cardiovascular abnormalities. E10.5 Tie2CrePten^flox/flox embryos showed an enlarged pericardial cavity, and frequent bleeding (red arrows) in the cavity (panels b,d,f) or in large trunk vessels (panel g). Panels e–g show H&E staining. (At) Atrium; (Ve) ventricle. (F) Decreased α-SMA expression in mutant embryonic vasculature (panels a–d) and heart (panels e,f). In E10.0 Tie2CrePten^+/+ embryos, perivascular walls in both the yolk sac (panel a) and dorsal aorta (panel c) are lined with α-SMA⁺ PCs/vSMCs. (Panels b,d) αSMA expression is dramatically reduced in E10.0 Tie2CrePten^flox/flox embryos. (Panels e,f) Reductions in the sizes of αSMA⁺ cardiac muscle walls, the intraventricular septum, and cardiac trabeculae were also observed in Tie2CrePten^flox/flox heart. (Panels g,h) Electron micrographs of E9.5 placentae reveal an absence of pericytes around the capillary network endothelium. (Blue arrows) Endothelial cells; (yellow arrows) blood cells in the vessel; (red arrows) pericytes.

Expression of VGFs, VGF receptors, and their downstream molecules. RT–PCR and protein analyses of expression of the indicated molecules important for cardiovascular development are shown. (A) RT–PCR of whole wild type (+/+) or Tie2CrePten^flox/flox (f/f) yolk sacs at E8.5. (B) RT–PCR of VEGFR2⁺ cells from E9.5 +/+ or f/f embryos. (C–E) RT–PCR (C), Western blotting (D), and flow cytometry (E) of HUVECs transfected with PTEN siRNA or mismatched control siRNA. Pten deficiency resulted in significantly reduced expression of connexin 40, Ang-1, ephrinB2, and VCAM-1 but significantly increased expression of Ang-2, VEGF-A, VEGFR1, VEGFR2, TGF-β1, and PAI-1. (F) RT–PCR of VEGFR2⁺ cells from E9.5 Tie2CrePten^+/+ (+/+) or Tie2CrePten^flox/+ (f/+) embryos that had been left untreated or stimulated with VEGF-A for 24 h. Pten heterozygosity in endothelial cells resulted in significantly increased expression of VEGF-A, VEGFR1, and VEGFR2. For A–F, data are representative of five independent experiments. GAPDH and actin served as loading controls.

Partial rescue of Pten deficiency by p110γ deficiency. (A,B) Mitigation of increased angiogenesis driven by VGFs in p110γ^-/-Tie2CrePten^flox/+ mice. (A) Representative photos of Matrigel plugs containing bFGF, bFGF + Ang-1, or bFGF + VEGF-A. Plugs were removed from mice of the indicated genotypes at 14 d post-injection. (B) Quantitated vascularization of the Matrigel plugs in A. p110γ deficiency significantly decreases the plug vascularization response (n = 6/genotype). (C,D) Mitigation of increased tumor growth. Shown are the results of anti-CD31 staining of sections of LLC tumors (C), quantitated tumor vascularization (D, left panel), and tumor growth (D, right panel) at 2 wk post-LLC inoculation (n = 4/genotype). Loss of p110γ decreased the tumor vascularization and tumor volume observed in Tie2CrePten^flox/+ mice. (E,F). Partial restoration of cardiovascular development in p110γ^-/-Tie2CrePten^flox/flox mice. Whole mounts stained with anti-CD31 antibody (E), and histological sections stained with α-SMA antibody (F) show that loss of p110γ dramatically rescues the growth retardation, vascular remodeling defect, reduction in vascular sprouting, enlarged pericardial cavity, and reduced thickness of the cardiac walls observed in Tie2CrePten^flox/flox mice. For B and D, quantitative results are expressed as the mean ± SEM and are representative of three trials. Statistical differences were determined using the Student's t-test; (*) p < 0.05.

Partial rescue of Pten deficiency by p85α deficiency. (A,B) Mitigation of increased angiogenesis driven by VGFs in p85a^-/-Tie2CrePten^flox/+ mice. (A) Representative photos of Matrigel plugs containing bFGF, bFGF + Ang-1, or bFGF + VEGF-A. Plugs were removed from mice of the indicated genotypes at 14 d post-injection. (B) Quantitated vascularization of the Matrigel plugs in A. p85α deficiency significantly decreases the plug vascularization response (n = 5/genotype). (C,D) Mitigation of increased tumor growth and tumor angiogenesis. Shown are the results of anti-CD31 staining of sections of LLC tumors (C), quantitated tumor vascularization (D, left), and tumor growth (D, right) at 2 wk post-LLC inoculation (n = 4/genotype). Loss of p85α decreased the tumor vascularization and tumor volume observed in Tie2CrePten^flox/+ mice. (E) Whole mounts of E10.5 and E14.5 embryos of the indicated genotypes showed that loss of p85α 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.