sPLA₂s induce the release of VEGF-A and VEGF-C from HLMs. A and B, HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (Control) or with the indicated concentrations of hGIIA, hGX, or LPS. VEGF-A (A) and VEGF-C (B) release was determined by ELISA. Data are mean ± SEM of four experiments. *p < 0.05 versus control. A, inset, HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (Ctr) or hGX (3 μg/ml). At the end of incubation, supernatants were concentrated by ultrafiltration and protein extracts (40 μg per sample) were immunoblotted with anti–VEGF-A Ab. rhVEGF-A₁₆₅ was used as positive control. Stripped membranes were reprobed with anti-GAPDH Ab to confirm equal protein loading. The Western blot shown is representative of three separate experiments. C, HLMs were incubated for the time indicated with RPMI 1640 alone [control, (○) and (□)] or with hGX [10 μg/ml, (●) and (■)]. VEGF-A [(○) and (●)] and VEGF-C [(□) and (■)] release was determined by ELISA. Data are mean ± SEM of four experiments. *p < 0.05 versus control.

sPLA₂-induced production of VEGF-A is independent from enzymatic activity and involves a receptor-mediated activation of HLMs. A, Effect of H48Q mutants of sPLA₂s on VEGF-A release. HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (ctr), with the wt hGIIA and hGX, or the mutants H48Q of hGIIA and hGX. VEGF-A release was determined by ELISA. Data are mean ± SEM of three experiments. *p < 0.05 versus control. B, Effect of Me-Indoxam and RO092906A on hGX-induced VEGF-A release. hGX (3 μg/ml) was preincubated (37°C, 15 min) with RPMI 1640 alone, or with the indicated concentrations of Me-Indoxam (●) or RO092906A (○). HLMs were then incubated (37°C, 24 h) with hGX (3 μg/ml) alone or with the various combinations of hGX with Me-Indoxam (●) or RO092906A (○). VEGF-A release was determined by ELISA. Data are expressed as percent inhibition of the maximum response induced by hGX alone calculated as (R – R_b)/(R_max – R_b) × 100, where R is the release in samples treated with the combination hGX plus inhibitor, R_b is the release in unstimulated samples, and R_max is the release in samples stimulated with hGX alone. Data are the mean ± SEM of three experiments. VEGF-A release induced by hGX alone was 661 ± 83 pg/mg protein. The unbroken and broken lines represent the best fit for inhibition of Me-Indoxam and RO092906A, respectively. C, Effect of MAPK inhibitors on sPLA₂-induced VEGF-A release. The cells were preincubated (37°C, 1 h) with SB203580 (30 μM), PD98059 (50 μM), or both (30 μM SB203580 and 50 μM PD988059) and then stimulated (37°C, 24 h) with hGX (3 μg/ml). VEGF-A release was determined by ELISA. Data are the mean ± SEM of three experiments. *p < 0.05 versus control; §p < 0.05 versus hGX alone.

Adenosine induces an angiogenic switch in sPLA₂-activated HLMs. A, Effect of NECA on hGX-induced release of VEGF-A. HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (ctr), NECA alone (0.1–10 μM), hGX alone (3 μg/ml), or the indicated combinations of hGX plus NECA. VEGF-A releasewas determined by ELISA. Data are the mean ± SEM of three experiments. *p < 0.05 versus control; ^§p < 0.05 versus hGX alone. B, Effect of NECA on hGX-induced release of TNF-α. HLMs were incubated (37°C, 24 h) as mentioned in A. TNF-α release was determined by ELISA. Data are the mean ± SEM of three experiments. *p < 0.05 versus control; ^§p < 0.05 versus hGX alone.

HLMs constitutively express different forms of VEGF. A, Expression of VEGF mRNAs. RNA extraction from resting HLMs and RT-PCR was performed as described under Materials and Methods. Specific RT-PCR amplification products for VEGFA (isoforms 189, 165, and 121), VEGFB (isoforms 186 and 167), VEGFC, VEGFD, PlGF, and GAPDH were separated on 2% agarose gel, stained with ethidium bromide, and visualized with an image analysis system. The experiment shown is representative of three separate experiments. B, Detection of VEGF proteins. HLM protein extracts (40 μg per sample) were immunoblotted with anti–VEGF-A (gel I and II), anti-PlGF (gel III), anti–VEGF-B (gel IV), anti–VEGF-C (gel V), and anti–VEGF-D (gel VI) Abs. rhVEGF-A₁₆₅, MCF-7 cells, EBNA expressing PlGF-1, RAW 264.7 cells were used as positive controls. Stripped membranes were reprobed with anti-GAPDH Ab to confirm equal protein content of each sample. Each Western blot shown is representative of three separate experiments.

Kinetics of hGX-induced mRNA expression of VEGFA₁₆₅ and VEGFC in HLMs. HLMs were incubated for the time indicated with RPMI 1640 alone or hGX (10 μg/ml). Real-time quantitative PCR was performed as described under Materials and Methods. The results were normalized for GAPDH. hGX-induced expression of VEGFA₁₆₅ (●) and VEGFC mRNA (○) was expressed as fold increase versus unstimulated cells. Data are mean ± SEM of three experiments. *p < 0.05 versus control.

Supernatants of HLMs activated by sPLA₂ induce an angiogenic response in the chick embryo CAM. Chick embryo CAMs at day 8 of incubation were implanted with gelatin sponges adsorbed with RPMI 1640 alone (A), supernatants of HLMs kept in culture for 24 h at 37°C with RPMI 1640 alone (control, B), with Me-Indoxam alone (10 μM, C), with hGX alone (3 μg/ml, D), or with the combination hGX plus Me-Indoxam (E). At day 12 of incubation, CAMs were examined as described under Materials and Methods. A–E, original magnification ×50. The experiment shown is representative of three separate experiments. In F, data are expressed as number of vessels at the sponge-CAM boundary and are the mean ± SEM of three experiments. *p < 0.05 versus control.

The angiogenic switch induced by adenosine in sPLA₂-activated HLMs involves a cooperation between A_2A and A₃ receptors. A, Effect of CGS21680, Cl-IB-MECA, and NECA on hGX-induced release of VEGF-A. HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (ctr), hGX alone (3 μg/ml), or combinations of hGX plus CGS21680 (0.1–10 μM), Cl-IB-MECA (0.1–10 μM), or NECA (10 μM). VEGF-A release was determined by ELISA. Data are the mean ± SEM of three experiments. *p < 0.05 versus control; ^§p < 0.05 versus hGX alone. B, Effect of CGS21680, Cl-IB-MECA, and NECA on hGX-induced release of TNF-α. HLMs were incubated (37°C, 24 h) as mentioned in A. TNF-α release was determined by ELISA. Data are the mean ± SEM of three experiments. *p < 0.05 versus control; ^§p < 0.05 versus hGX alone.