Aikaterini Detoraki, MD, PhD, Rosaria I

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Vascular endothelial growth factors synthesized by human lung mast cells exert angiogenic effects Aikaterini Detoraki, MD, PhD, Rosaria I. Staiano, PhD, Francescopaolo Granata, MD, PhD, Giorgio Giannattasio, MD, Nella Prevete, PhD, Amato de Paulis, MD, Domenico Ribatti, MD, Arturo Genovese, MD, Massimo Triggiani, MD, PhD, Gianni Marone, MD Journal of Allergy and Clinical Immunology Volume 123, Issue 5, Pages 1142-1149.e5 (May 2009) DOI: 10.1016/j.jaci.2009.01.044 Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Expression of VEGFs in human mast cells. A, VEGFA, VEGFB, VEGFC, VEGFD, and PlGF RT-PCR amplification products from HLMCs, LAD-2 cells, and HMC-1 cells. B, Immunoblot with anti–VEGF-B, anti–VEGF-C, anti–VEGF-D, and anti–GAPDH antibodies of HLMC, LAD-2, and HMC-1 lysates. MCF-7 and RAW 264.7 (Raw) cells were used as positive controls. Data are representative of 4 experiments. Journal of Allergy and Clinical Immunology 2009 123, 1142-1149.e5DOI: (10.1016/j.jaci.2009.01.044) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Effects of PGE2 on VEGF production from human mast cells. A, LAD-2 cells were incubated (3-9 hours) with PGE2 (10−6 mol/L). Expression of VEGFA165, VEGFB167, VEGFC, and VEGFD was determined by using qPCR. Data are expressed as fold increase versus unstimulated cells and are the mean ± SE of 3 experiments. B, HLMCs were incubated (3-48 hours) with medium alone (control) or PGE2 (10−6 mol/L). VEGF-A release was determined by using ELISA. Data are the mean ± SE of 3 experiments. ∗P < .01 vs control. Journal of Allergy and Clinical Immunology 2009 123, 1142-1149.e5DOI: (10.1016/j.jaci.2009.01.044) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Effects of NECA on VEGF production from human mast cells. A, HMC-1 cells were incubated (3-9 hours) with NECA (10−5 mol/L). Expression of VEGFA165, VEGFB167, VEGFC, and VEGFD was determined by using qPCR. Data are expressed as fold increase versus unstimulated cells and are the mean ± SEM of 3 experiments. B, HLMCs were incubated (3-24 hours) with medium alone (control) or NECA (10−5 mol/L) in the presence of adenosine deaminase (1 U/mL). VEGF-A release was determined by using ELISA. Data are the mean ± SEM of 3 experiments. ∗P < .01 versus control. Journal of Allergy and Clinical Immunology 2009 123, 1142-1149.e5DOI: (10.1016/j.jaci.2009.01.044) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Angiogenic activity of HLMC supernatants in the CAM assay. Representative experiment of 3 in which CAMs were implanted with sponges containing medium alone (A), supernatants of HLMCs incubated without (B) or with PGE2 (C, 10−6 mol/L) or NECA (D, 10−5 mol/L). In E and F supernatants from PGE2- or NECA-activated HLMCs, respectively, were pretreated with anti-VEGF-A antibody. Journal of Allergy and Clinical Immunology 2009 123, 1142-1149.e5DOI: (10.1016/j.jaci.2009.01.044) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Expression of VEGFRs in human mast cells. A, VEGFR-1, soluble VEGFR-1 (sVEGFR-1), VEGFR-2, and GAPDH RT-PCR amplification products from HLMCs, LAD-2 cells, and HMC-1 cells. B, HLMCs were incubated (at 4°C for 20 minutes) with fluorescein isothiocyanate–labeled anti-IgE, PE-labeled anti–VEGFR-1, PE-labeled anti–VEGFR-2, or isotype-matched antibodies. Flow cytometry was performed as described in the Methods section. Data are representative of 4 experiments. Journal of Allergy and Clinical Immunology 2009 123, 1142-1149.e5DOI: (10.1016/j.jaci.2009.01.044) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Effects of VEGFs on chemotaxis of HLMCs. A, HLMC chemotaxis was induced by incubation (at 37°C for 3 hours) with VEGF-A165, VEGF-B167, VEGF-C, VEGF-D, and PlGF. Data are the mean ± SEM of 3 experiments. ∗P < .01 versus the respective concentration of VEGF-A165, VEGF-C, and VEGF-D. B, HLMCs were preincubated (at 37°C for 30 minutes) with anti–VEGFR-1 antibody, anti–VEGFR-2 antibody, a combination of both antibodies, or irrelevant mouse antibody. The cells were washed twice with PBS, and chemotaxis was then induced by means of incubation (at 37°C for 3 hours) with VEGF-A165. Data are the mean ± SEM of 3 experiments. ∗P < .01 versus VEGF-A165 alone. ∗∗P < .01 versus anti–VEGFR-1 plus VEGF-A165. Journal of Allergy and Clinical Immunology 2009 123, 1142-1149.e5DOI: (10.1016/j.jaci.2009.01.044) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Effects of PGE2 on VEGF expression in human mast cells Effects of PGE2 on VEGF expression in human mast cells. LAD-2 cells were incubated (at 37°C for 3 hours for VEGF-A and VEGF-C and 6 hours for VEGF-B and VEGF-D) without or with increasing concentrations (10−9 to 10−6 mol/L) of PGE2. Expression of VEGF-A165, VEGF-B167, VEGF-C, and VEGF-D was determined by means of real-time qPCR (see the Methods section). Data are expressed as fold increase versus unstimulated cells and are the mean ± SEM of 3 experiments. ∗P < .05 versus unstimulated. Journal of Allergy and Clinical Immunology 2009 123, 1142-1149.e5DOI: (10.1016/j.jaci.2009.01.044) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Effects of NECA on VEGF expression in human mast cells Effects of NECA on VEGF expression in human mast cells. HMC-1 cells were incubated (at 37°C for 9 hours) without or with increasing concentrations (10−8 to 10−5 mol/L) of NECA. Expression of VEGF-A165, VEGF-B167, VEGF-C, and VEGF-D was determined by means of real-time qPCR (see the Methods section). Data are expressed as fold increase versus unstimulated cells and are the mean ± SEM of 3 experiments. ∗P < .05 versus unstimulated. Journal of Allergy and Clinical Immunology 2009 123, 1142-1149.e5DOI: (10.1016/j.jaci.2009.01.044) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Effects of VEGF-A165 on VEGFR expression in human mast cells Effects of VEGF-A165 on VEGFR expression in human mast cells. HLMCs were incubated (at 37°C for 0, 1, and 3 hours) without (Control) and with VEGF-A165 (250 ng/mL). At each time point, flow cytometry for VEGFR-1 (A) and VEGFR-2 (B) was performed as described in the Methods section. Data are expressed as the net mean fluorescence intensity measured in 3 different experiments and represent the mean ± SEM. Journal of Allergy and Clinical Immunology 2009 123, 1142-1149.e5DOI: (10.1016/j.jaci.2009.01.044) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions