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First published online 30 March 2004
doi: 10.1242/jcs.01061

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EphA2 receptor tyrosine kinase regulates endothelial cell migration and vascular assembly through phosphoinositide 3-kinase-mediated Rac1 GTPase activation

¹ Division of Rhematology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
² Division of Nephrology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
³ Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
⁴ Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
⁵ Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
⁶ Department of Chemistry, Maryville College, Maryville, Tennessee 37801, USA
⁷ Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202 USA

View larger version (15K): [in a new window]   Fig. 1. Targeted disruption of the mouse ephA2 gene. (A) Map of ephA2 targeting vector, ephA2 and recombined loci. PT1.ephA2 consists of a 2.8 kb EcoRI fragment (3' homology) flanking a pMCIneo expression cassette. The black box represents ephA2 exonic sequences that are disrupted by the neo cassette. The gray box between the EcoRI and SacI sites in the genomic locus map represents a unique sequence located outside the targeting vector sequences used to detect homologous recombination events by Southern blot. B, BglII; H, HindIII; R, EcoRI; X, XbaI. (B) Southern blot analysis of tail DNA confirmed the presence of the wild-type allele in wild-type (+/+) and heterozygous (+/–) mice and the presence of the targeted allele in heterozygous (+/–) and null (–/–) mice.

View larger version (62K): [in a new window]   Fig. 2. EphA2-deficiency impairs ephrin-A1-induced vascular assembly in vitro. (A) Lung microvascular endothelial cells (MPMEC) isolated from wild-type (+/+), heterozygous (+/–), or EphA2-deficient (–/–) mice were plated on a thin layer of growth-factor-reduced Matrigel in the presence or absence of ephrin-A1 to examine and quantify vascular assembly. After 9 hours, the endothelial cells were photographed. Scale bar, 20 µm. (B) The numbers of intersections between endothelial cell branches were counted. Four fields per culture were scored for each condition and data are means±s.d. of three independent experiments. Significant differences in assembly for EphA2-/– cells stimulated with ephrin-A1 (*) compared to other experimental conditions are indicated for P<0.01 using ANOVA analysis. (C) EphA2-deficient (–/–) MPMEC were transduced with recombinant adenoviruses encoding wild-type EphA2 (Ad-EphA2) or control ß-galactosidase (Ad-ßgal). After 48 hours, the cells were plated on a thin layer of growth factor-reduced Matrigel in the presence or absence of ephrin-A1 for vascular assembly assay and photographed after 9 hours. Scale bar, 20 µm. For MPMEC (–/–) Ad-EphA2, the left hand panels show bright-field images, and the right hand panels show fluorescence images of identical fields displaying co-expression of GFP from the adenovirus plasmid. (D) The numbers of intersections between endothelial cell branches were counted. Four fields per culture were scored for each condition and data are presented as means±s.d. of three independent experiments. Significant differences in assembly are indicated where P<0.01 (using Student's t-test) for EphA2-/– Ad-EphA2 (**) versus EphA2-/– Ad-ßgal and for EphA2-/– Ad-EphA2 + ephrin-A1 (***) versus EphA2-/– Adßgal + ephrin-A1. (E) Immunoblot analysis of ßgal or EphA2 expression in lysates from MPMEC transduced with recombinant adenoviruses.

View larger version (26K): [in a new window]   Fig. 3. EphA2 receptor is required for ephrin-A1-induced endothelial cell migration. (A) Immunoblot analysis of EphA2 expression in wild-type (+/+), heterozygous (+/–) and Ad-EphA2 transduced EphA2-/– MPMEC lysates versus Ad-ßgal transduced EphA2-/– MPMEC lysates. (B) Migration of MPMEC derived from wild-type, heterozygous, or EphA2-deficient mice in response to ephrin-A1 was quantified by transwell assay. EphA2-deficient MPMECs were infected with recombinant adenoviruses encoding ß-galactosidase (EphA2–/–Ad-ßgal) or wild-type EphA2 (EphA2–/–Ad-EphA2) 48 hours prior to migration assay. The number of endothelial cells that had migrated to the lower surface of the transwell were counted. Three fields per transwell were scored for each condition in triplicate samples and data are means±s.d. of three independent experiments. Significant differences in migration for EphA2–/–Ad-EphA2 (*) or Ad-ßgal (**) compared to other experimental conditions are indicated where P<0.01 using ANOVA analysis. (C) Immunoblot analysis of EphA2 immunoprecipitated from BPMEC lysates showing elevated tyrosine phosphorylation of EphA2-NeuTM mutant in the absence of ephrin-A1 stimulation. (D) Immunoblot analysis of immunoprecipitated EphA2 showing decreased tyrosine phosphorylation of endogenous EphA2 after ephrin-A1 stimulation in EphA2-C-expressing BPMEC relative to mock transfected cells. (E) Migration of BPMEC expressing kinase elevated EphA2 (EphA2-NeuTM) or truncated, dominant negative EphA2 (EphA2-C) in response to ephrin-A1 was also quantified by transwell assay. Significant differences in migration are indicated for P<0.01 using Student's t-test: ***P=0.0001 EphA2-NeuTM +/–ephrin-A1 versus mock unstimulated, ****P=0.0005 EphA2-C + ephrin-A1 versus mock + ephrin-A1. (F) Expression of EphA2-NeuTM or EphA2-C in BPMEC was confirmed by immunoblot analysis.

View larger version (31K): [in a new window]   Fig. 4. EphA2-mediated endothelial cell migration is regulated by Rac1 GTPase. (A) Active GTP-bound forms of Rac1 and cdc42 were analyzed by Pak-PBD pulldown followed by immunoblot in lysates from heterozygous (+/–) or EphA2-deficient (–/–) MPMEC stimulated with ephrin-A1. Total Rac1 and cdc42 levels within the lysate prior to PBD-pulldown were detected by immunoblot. Data are representative of four independent experiments. (B) Activation of Rac1 induced by ephrin-A1 stimulation was confirmed in BPMEC, and occurred upon initiation of EphA2 autophosphorylation. (C) Ephrin-A1-induced migration of mock transfected or EphA2-NeuTM-expressing BPMEC in the presence or absence of GTPase inhibitor Toxin B was quantified by transwell assay. Significant differences in migration are indicated where P<0.01 using Student's t-test: *P=0.0001 mock + ephrin-A1 versus mock + ephrin-A1/Toxin B, **P=0.0005 EphA2-NeuTM versus EphA2-NeuTM/Toxin B. (D) Migration of BPMEC transfected with an expression construct encoding dominant negative Rac1 (Rac1-N17) in response to ephrin-A1 was also quantified by transwell assay by scoring three fields per transwell for each condition in triplicate samples. Data are means±s.d. of three independent experiments. Significant differences in migration are indicated where P<0.01 using Student's t-test: ***P=0.0005 mock + ephrin-A1 versus Rac1-N17 + ephrin-A1. Expression of Rac1-N17 was confirmed by myc immunoblot.

View larger version (30K): [in a new window]   Fig. 5. EphA2-mediated Rac1 activation and migration is dependent on PI3K. (A) Active GTP-bound form of Rac1 was analyzed by Pak-PBD pull-down assay using lysates from ephrin-A1-stimulated BPMEC in the presence or absence of PI3K inhibitors, wortmannin or LY294002. Total Rac1 levels within the lysate prior to PBD-pulldown were detected by immunoblot. Data are representative of three independent experiments. (B) Migration of ephrin-A1-stimulated BPMEC, in the presence or absence of LY294002 PI3K inhibitor, was quantified by transwell assay by scoring three fields per transwell for each condition in triplicate samples. Data are means±s.d. of three independent experiments. Significant differences in migration are indicated where P<0.01 using Student's t-test: *P=0.0005 vehicle + ephrin-A1 versus LY294002 + ephrin-A1. (C) Activation of Rac1 was determined by Pak-PBD pull-down assay in BPMEC expressing a dominant negative p85 PI3K regulatory subunit (-p85) in response to ephrin-A1 stimulation. (D) Migration of BPMEC transfected with -p85 in response to ephrin-A1 was quantified by transwell assay by scoring three fields per transwell for each condition in triplicate samples. Data are means±s.d. of three independent experiments. Significant differences in migration are indicated where P<0.01 using Student's t-test: **P=0.0005 mock + ephrin-A1 versus -p85 + ephrin-A1. Expression of -p85 was confirmed by immunoprecipitation of p85 (endogenous and truncated -p85) followed by p85 immunoblot.

View larger version (84K): [in a new window]   Fig. 6. EphA2-deficiency impairs ephrin-A1-induced angiogenesis in vivo. (A) Sponges impregnated with ephrin-A1 or IgG were subcutaneously implanted into the dorsal flank of EphA2 heterozygous (EphA2+/–) or EphA2-deficient (EphA2–/–) mice. After 7 days, mice were injected intravenously with TRITC-dextran to visualize host blood vessels associated with sponges. Fewer surface vessels were associated with ephrin-A1-treated sponges in EphA-/– animals relative to EphA+/– controls. Scale bar, 5 mm. Arrowheads indicate surface blood vessels covering sponges. (B) Sponge sections were counterstained with DAPI to visualize nuclei relative to TRITC vessel labeling. Vessel infiltration in the ephrin-A1-treated sponge periphery was detected in control EphA+/– animals, but not in EphA-/– animals. Scale bar, 10 µm. Dashed line indicates the boundary between adjacent host skin tissue (left) and sponge (right). Arrows indicate TRITC-positive vessels that have infiltrated into the sponge, and (*) indicate vessels within the host skin tissue. (C) Left panel displays higher magnification (40x) of the upper left panel in B. Scale bar, 5 µm. Right panel displays low magnification (10x) of the lower left panel in B, demonstrating the distance of host vessels relative to the boundary of IgG-treated sponges in EphA+/– animals. Scale bar, 1 mm. Data are a representative of results from three independent mice per genotype.

View larger version (80K): [in a new window]   Fig. 7. EphA2-deficiency impairs vascular assembly in vivo. (A) Lung microvascular endothelial cells (MPMEC) isolated from heterozygous (+/–) or EphA2-deficient (–/–) mice were transduced with adenoviruses encoding nuclear ß-galactosidase, suspended in growth-factor-reduced Matrigel, and transplanted into nude mice to examine vascular assembly in vivo. (A) After 4 and 10 days, Matrigel plugs were collected and cryosections were X-gal stained to detect exogenous endothelial cells. Scale bar, 10 µm. Arrowheads indicate LacZ-positive exogenous MPMEC. (B) Assembled vascular structures were detected in EphA2+/– plugs, but not in EphA2-/– plugs, after 7 days. Scale bar in upper panels, 10 µm; lower panels, 5 µm. Arrowheads indicate LacZ-positive exogenous MPMEC. No LacZ-positive cells were detected in plugs containing Matrigel only. (C) Cryosections from MPMEC plugs were co-stained for LacZ expression and CD31. Both heterozygous and EphA2-deficient MPMEC were positive for CD31 staining (arrows), though EphA2-deficient MPMEC lacked the elongated, endothelial morphology observed in heterozygous MPMEC. LacZ-positive, exogenous MPMEC from heterozygous mice associated with host endothelium (asterisk), while EphA2-deficient MPMEC did not. Scale bar, 2 µm. Data are representative of six independent plugs per genotype derived from three independent donor mice from each genotype.

View larger version (38K): [in a new window]   Fig. 8. EphA2-deficiency impairs endothelial cell survival in vivo. (A) Matrigel plugs harboring MPMEC isolated from heterozygous (+/–) or EphA2-deficient (–/–) mice were collected 4 days (not shown) or 10 days post-transplantation and co-stained for LacZ expression and the proliferation marker Ki67. Arrows indicate LacZ+/Ki67+ nuclei, and the asterisks indicate LacZ+/Ki67–nuclei. Scale bar, 2.5 µm. (Right) The percentage of LacZ+/Ki67+ nuclei relative to total LacZ+ nuclei in each field was calculated to quantify proliferation in exogenous endothelial cells. (B) Matrigel plugs were also subjected to staining for LacZ followed by TUNEL assay to detect apoptosis at 10 days (left). Arrows indicate LacZ+/TUNEL+ nuclei, and the asterisks indicate LacZ+/TUNEL–nuclei. Scale bar, 5 µm. (Right) The percentage of LacZ+/TUNEL+ nuclei relative to total LacZ+ nuclei in each field was calculated to quantify apoptosis in exogenous endothelial cells. Data are means±s.d. of three independent samples/condition. Significant differences in percentage of apoptotic nuclei are indicated where P<0.01 using Student's t-test: *P=0.003 EphA2+/– versus EphA2-/– 4-day plugs, **P=0.03 EphA2+/– versus EphA2-/– 10-day plugs.

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