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First published online 18 December 2002
doi: 10.1242/jcs.00286

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VEGF expression in human macrophages is NF-B-dependent: studies using adenoviruses expressing the endogenous NF-B inhibitor IB and a kinase-defective form of the IB kinase 2

Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London W6 8LH, UK

View larger version (31K): [in a new window]   Fig. 1. Inhibition of p38MAPK, p42/44 MAPK and PI3-kinase pathways partially suppress LPS-induced VEGF production by macrophages. (A) Comparison of the kinetics of LPS-induced VEGF and TNF- production. Macrophages at 1x106 cells/ml were stimulated with 10 ng/ml LPS and, at the indicated times, culture supernatants were harvested to assay production of VEGF and TNF- by ELISA. In subsequent experiments macrophages were pre-incubated with the indicated doses of either SB203580 (B), PD098059 (C), wortmannin (D) or LY294002 (E) for 1 hour before stimulating with 10 ng/ml LPS. After 24 hours of culture, media were collected and VEGF was measured by ELISA. The mean±s.d. of triplicate wells is shown. Data are representative of three experiments: ***P<0.001, **P<0.01 versus cells stimulated with LPS alone.

View larger version (30K): [in a new window]   Fig. 2. Overexpression of IB in M-CSF-differentiated human macrophages inhibits LPS-induced NF-B activation and VEGF production. Macrophages were either left uninfected or were infected for 2 hours in serum-free medium with control adenovirus, or an adenovirus expressing IB. Cells were cultured in 5% FCS for 2 days and subsequently stimulated with 10 ng/ml LPS for 45 minutes. Cytosolic and nuclear extracts were then collected and examined for cytosolic levels of IB (A) and -tubulin (B) by western blot analysis or for NF-B translocation by EMSA (C). A representative of three independent experiments is shown. For VEGF production, macrophages were left uninfected, or infected for 2 hours in serum-free medium with control adenovirus or with an adenovirus-expressing IB. After 2 days of further culture, cells were stimulated with 10 ng/ml LPS for 24 hours. Supernatants were collected and assayed by ELISA (D). Data are means±s.d. of culture triplicates, and are representative of ten different experiments: ***P<0.001 versus response with control adenovirus-infected and uninfected cells.

View larger version (30K): [in a new window]   Fig. 3. Expression of IKK-2dn in human macrophages does not inhibit LPS-induced NF-B activation but partially decreases VEGF production. Macrophages were either left uninfected, or were infected for 2 hours in serum-free medium with control adenovirus, or adenoviruses expressing IKK-1dn and IKK-2dn. Cells were cultured in 5% FCS for 2 days and stimulated with 10 ng/ml LPS for 45 minutes. Cytosolic and nuclear extracts were then collected and examined for cytosolic levels of IKK-2 (A), IB (B) and -tubulin (C) by western blot analysis or for NF-B translocation by EMSA (D). A representative of three independent experiments is shown. For VEGF production, macrophages were left uninfected, or were infected for 2 hours in serum-free medium with control adenovirus or with an adenovirus expressing IKK-2dn. After 2 days of further culture, cells were stimulated with 10 ng/ml LPS for 24 hours (E). Data are expressed as means±s.d. of triplicates and are representative of ten different experiments: *P<0.05 versus response with control adenovirus-infected and uninfected cells.

View larger version (24K): [in a new window]   Fig. 4. Expression of IB and IKK-2dn inhibits CD40L-induced VEGF release by macrophages. (A) M-CSF-differentiated macrophages were cultured for 24 hours in 30 mm2 wells with 5x105 CD40L-transfected (CD40L+) or control (MOCK) cells. After 24 hours, supernatants were collected and assayed by ELISA for VEGF. In parallel experiments, macrophages were left uninfected, or infected for 2 hours in serum-free medium with Advß-gal, AdvIKK-2dn and AdvIB, at m.o.i. 100:1. Cells were cultured for a further 2 days and then stimulated with 1 µg/ml sCD40L. After 45 minutes, cytosolic and nuclear extracts were obtained and examined for the presence of IKK-2dn (B) and IB (C) by western blotting. A representative of three independent experiments is shown. Equal amounts of protein were loaded, as determined by re-probing for -tubulin (not shown). For measurement of VEGF production, macrophages were left uninfected, or infected as described above and after 2 days stimulated with CD40L-transfected (CD40L+) or (MOCK) cells as control. After 24 hours, supernatants were collected and assayed for VEGF (D). Mean cytokine production±s.d. of triplicate cultures is shown and is representative of three independent experiments: **P<0.01, ***P<0.001 versus response with control adenovirus-infected and uninfected cells.

View larger version (15K): [in a new window]   Fig. 5. Endogenous TNF- is required for LPS- and CD40L-induced macrophage VEGF release. Macrophages were stimulated for 24 hours with either LPS (10 ng/ml) or sCD40L (1 µg/ml) in the presence of the isotype control antibody IgG2 10 µg/ml or the anti-TNF- antibody (cA2) 10 µg/ml. Culture supernatants were assayed for VEGF. Neutralisation of endogenous TNF- significantly inhibits VEGF production by macrophages in response to LPS (A) and sCD40L (B). Mean cytokine production±s.d. of triplicate cultures is shown and is representative of three independent experiments.

View larger version (13K): [in a new window]   Fig. 6. Possible NF-B-like binding sites within the VEGF 5'-promoter region. Sequences that differ at the level of one or two positions from the decameric B-like binding sites (5'-HGGARNYYCC-3', where H indicates A, C, or T; R indicates A or G; Y indicates C or T; N indicates any nucleotide) and NF-B-like consensus sequence (5'-GGGRNNYYCC-3', where R indicates A or G, Y indicates C or T, and N indicates any nucleotide) in the VEGF promoter (Tischer et al., 1991) are highlighted (bold and underlined).