Cytokine and estrogen stimulation of endothelial cells augments activation of the prekallikrein-high molecular weight kininogen complex: Implications.

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Cytokine and estrogen stimulation of endothelial cells augments activation of the prekallikrein-high molecular weight kininogen complex: Implications for hereditary angioedema Kusumam Joseph, PhD, Baby G. Tholanikunnel, PhD, Allen P. Kaplan, MD Journal of Allergy and Clinical Immunology Volume 140, Issue 1, Pages 170-176 (July 2017) DOI: 10.1016/j.jaci.2016.09.032 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Hsp90 binding to HK and prekallikrein. Increasing concentrations of HK (A) and prekallikrein (B) were incubated in Hsp90 (5 μg/mL)-coated plates for 1 hour and probed with polyclonal antibodies to each followed by secondary antibody and color development. The binding was performed in the presence or absence of 50 μM zinc chloride. Each concentration was in duplicate and the assay was performed at 3 times. Journal of Allergy and Clinical Immunology 2017 140, 170-176DOI: (10.1016/j.jaci.2016.09.032) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Hsp90 release from activated endothelial cells. Cells were grown in 24-well plate with 0.5 mL medium. When confluent, the cells were washed and changed to serum-free medium for treatment with bradykinin, IL-1-β, TNF-α, and estradiol and incubated at 37°C. A, Dose-response: Cells were treated for 2 hours with varying concentrations of each compound as indicated and the supernatant was collected and assayed by ELISA. The graph represents mean ± SD of 3 separate experiments. Values that are significantly higher than control are indicated with asterisks (*P < .05). B, Time course: Samples were collected at indicated times and the levels of Hsp90 released were measured by ELISA. The graph represents mean ± SD of 3 separate experiments. *Indicate values that are significantly higher than control (P < .05) at a comparable time point. Journal of Allergy and Clinical Immunology 2017 140, 170-176DOI: (10.1016/j.jaci.2016.09.032) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Kallikrein formation on stimulated human umbilical vein endothelial cells. Cells were grown in 96-well plate with 0.2 mL medium. When confluent, the cells were washed and changed to serum-free medium and treated with IL-1-β (B), TNF-α (C), and estradiol (D) for 1 hour before the addition of HK, prekallikrein (PK), and factor XII (FXII). The kallikrein activity was measured using a chromogenic substrate (S2302, Diapharma). Kallikrein formation on untreated control cells is shown in A. The experiments were performed 3 times with similar results and a representative curve is shown. Journal of Allergy and Clinical Immunology 2017 140, 170-176DOI: (10.1016/j.jaci.2016.09.032) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Factor XII activation on endothelial cells in the presence of prekallikrein and HK. The experiments were performed 3 times with similar results and a representative curve is shown. Journal of Allergy and Clinical Immunology 2017 140, 170-176DOI: (10.1016/j.jaci.2016.09.032) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Urokinase and tissue plasminogen activator release from endothelial cells. Cells were grown in 24-well plate with 0.5 mL medium. When confluent, the cells were washed and changed to serum-free medium for treatment with thrombin, bradykinin, IL-1-β, TNF-α, and estradiol and incubated at 37°C. Samples were collected at indicated times and the levels of urokinase (A) and TPA (B) released was measured by ELISA. The experiments were performed 3 times with duplicate wells, and the graphs represent mean ± SD. *Indicate values that are significantly higher than control (P < .001). Journal of Allergy and Clinical Immunology 2017 140, 170-176DOI: (10.1016/j.jaci.2016.09.032) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Factor XII activation by kallikrein vs plasmin. Factor XII at 2.5 μg/mL was incubated at 37°C in the presence of increasing concentration of kallikrein (A) and plasmin (B) and factor XIIa activity was measured using a specific synthetic substrate (glu-gly-arg p-nitroanilide). The experiments were performed 3 times with similar results and a representative curve is shown. Journal of Allergy and Clinical Immunology 2017 140, 170-176DOI: (10.1016/j.jaci.2016.09.032) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions