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First published online 16 December 2003
doi: 10.1242/jcs.00871

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Integrin-linked kinase regulates vascular morphogenesis induced by vascular endothelial growth factor

Cancer Research Laboratory, Taiho Pharmaceutical Co. Ltd, 1-27 Misugidai, Hanno, Saitama, 357-8527, Japan

View larger version (47K): [in a new window]   Fig. 1. Effects of ILK-KD and LY294002 on morphological changes induced by VEGF. (A) HUVEC (1.5x104 cells/well) were incubated for 8 hours in serum-free medium in the presence of the indicated concentration of rhVEGF. The morphological changes of HUVEC on collagen type I gel were evaluated as described in Materials and Methods. (B) HUVEC were transiently transfected with the indicated plasmids 18 hours before VEGF stimulation. LY294002 was added 30 minutes before the stimulation. The cells were incubated in serum-free medium in the presence of 30 ng/ml rhVEGF and the morphological changes were examined. Data are expressed as the mean ± s.d. (C) Images showing typical morphological changes of HUVEC.

View larger version (12K): [in a new window]   Fig. 2. VEGF increased adhesion of HUVEC to collagen type I. (A) HUVEC were starved for 18 hours and then plated on 96-well plates (5.0x104 cells/well). VEGF was added to the medium 10 minutes before plating and the adherent cells were measured by WST-1 assay, as described in Materials and Methods. (B) HUVEC were transiently transfected with the indicated plasmids 18 hours before VEGF stimulation. LY294002 was added 30 minutes before the stimulation. Data are expressed as the mean ± s.d.

View larger version (35K): [in a new window]   Fig. 3. PKB/Akt phosphorylation on serine 473, induced by VEGF, was abrogated by ILK inhibition. (A) VEGF-induced phosphorylation of PKB/Akt. HUVEC and LNCaP were stimulated with 30 ng/ml of rhVEGF and 100 ng/ml of rhEGF, respectively, for 15 minutes. (B) Inhibition of serine 473 phosphorylation of PKB/Akt by ILK-KD or ILK-AS. The cells transfected with the indicated plasmids were stimulated with 30 ng/ml of rhVEGF for 15 minutes, and then PKB/Akt phosphorylation on serine 473 was detected by immunoblot analysis. (C) VEGF increased kinase activity of ILK. The cells were lysed, and ILK was immunoprecipitated from cell extracts. ILK activity was determined using PKB/Akt as a substrate. Anti-ILK immunoblot was prepared from the same immunoprecipitates used for the kinase assay (top panel). (D) Inhibition of kinase activity of ILK by ILK-KD or ILK-AS. The cells transfected with the indicated plasmids were stimulated with 30 ng/ml of rhVEGF for 5 minutes, and then ILK activity was measure by ILK kinase assay. Anti-ILK immunoblot was prepared from the same immunoprecipitates used for the kinase assay (top panel).

View larger version (23K): [in a new window]   Fig. 4. VEGF stimulates association of ILK with VEGFR complex. (A) VEGF stimulates recruitment of ILK to VEGFR-2. The cells were cultured in the presence or absence of 30 ng/ml of rhVEGF for 5 minutes. The cells were lysed, and anti-VEGFR-2 immunoprecipitates were prepared and immunoblotted with anti-VEGFR-2, anti-phosphotyrosine or anti-ILK antibodies. (B) Inhibition of kinase activity of ILK by LY294002. The cells were stimulated with 30 ng/ml of rhVEGF for 5 minutes, and then ILK activity was measure by ILK kinase assay. LY294002 was added 30 minutes before the VEGF stimulation. Anti-ILK immunoblot was prepared from same immunoprecipitates used for the kinase assay (top panel).

View larger version (27K): [in a new window]   Fig. 5. Effect of ILK-AS on the apoptosis-suppressive activity of VEGF. HUVEC were transiently transfected with the indicated plasmids and incubated for 24 hours. Apoptosis was then induced by serum starvation in the presence or absence of 30 ng/ml of rhVEGF for 48 hours. The cells were then harvested, and apoptotic cell death was determined by flow cytometry analysis using annexin-V-fluorescein. One of two comparable experiments is shown.

View larger version (23K): [in a new window]   Fig. 6. Effect of ILK-KD and ILK-AS on VEGF-mediated suppression of caspase-3/7 activities. HUVEC were transiently transfected with the indicated plasmids and incubated for 24 hours. Caspase-3/7 activation was then induced by serum starvation in the presence or absence of 30 ng/ml of rhVEGF for 48 hours. Caspase-3/7 activities were measured by Apo-ONETM Homogeneous caspase-3/7 assay, as described in Materials and Methods.

View larger version (16K): [in a new window]   Fig. 7. Effect of ILK-AS on HUVEC proliferation stimulated with VEGF. HUVEC were transiently transfected with the indicated plasmids and incubated for 16 hours. The cells were cultured in the presence or absence of 30 ng/ml of rhVEGF for 48 hours. Viable cells were measured by WST assay. Data are expressed as the mean ± s.d.

View larger version (13K): [in a new window]   Fig. 8. Effects of ILK-KD and LY294002 on chemotactic migration of HUVEC stimulated by VEGF. HUVEC were transiently transfected with the indicated plasmids 18 hours before VEGF stimulation. LY294002 was added to the upper chamber containing HUVEC 30 minutes before the stimulation. rhVEGF was added to the lower chamber at a concentration of 30 ng/ml and migratory activity of the cells was estimated, based on the number of cells migrating to the lower chamber. Data are expressed as the mean ± s.d.

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