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An antibody against HK blocks Alzheimer's disease peptide β-amyloid-induced bradykinin release in human plasma - PubMed

  • ️Tue Jan 01 2019

An antibody against HK blocks Alzheimer's disease peptide β-amyloid-induced bradykinin release in human plasma

Zu-Lin Chen et al. Proc Natl Acad Sci U S A. 2019.

Abstract

Bradykinin is a proinflammatory factor that mediates angioedema and inflammation in many diseases. It is a key player in some types of hereditary angioedema and is involved in septic shock, traumatic injury, Alzheimer's disease (AD), and stroke, among others. Activation of the plasma contact system leads to elevated levels of plasma kallikrein, which cleaves high molecular weight kininogen (HK) to release bradykinin. Drug development for bradykinin-meditated pathologies has focused on designing inhibitors to the enzymes that cleave HK (to prevent bradykinin release) or antagonists of endothelial bradykinin receptors (to prevent downstream bradykinin action). Here we show a strategy to block bradykinin generation by using an HK antibody that binds to HK, preventing its cleavage and subsequent bradykinin release. We show that this antibody blocks dextran sodium sulfate-induced HK cleavage and bradykinin production. Moreover, while the pathogenic AD peptide β-amyloid (Aβ)42 cleaves HK and induces a dramatic increase in bradykinin production, our HK antibody blocked these events from occurring. These results may provide strategies for developing treatments for bradykinin-driven pathologies.

Keywords: Alzheimer’s disease; beta-amyloid; bradykinin; high molecular weight kininogen.

Copyright © 2019 the Author(s). Published by PNAS.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.

HK antibodies bind HK and block DXS-induced HK cleavage and bradykinin production in human plasma. (A) Human plasma was incubated with biotinylated 3E8, 2B7, and 4B12 HK antibodies and control IgG, and streptavidin was added to pull down the antibody−antigen complex. The samples were analyzed by Western blot using a commercial HK antibody. The 3E8 and 2B7 HK antibodies bound HK and immunoprecipitated it from human plasma, while 4B12 HK antibody and control IgG did not pull down HK from human plasma. (B) HK Western blot showed 3E8 HK antibody dose-dependently blocked DXS-induced HK cleavage. Without HK antibodies, DXS induced complete cleavage of HK (disappearance of HK band and appearance of cHK band). The 2B7 and 4B12 HK antibodies and control IgG did not show any effects on HK cleavage. (C) The protective effects of 3E8 antibody were quantified by measuring the intensity of HK bands after normalization to TF. The 3E8 at a higher concentration (3.75 μM) completely blocked DXS-induced HK cleavage, and 3E8 at lower concentration (0.75 μM) also significantly blocked HK cleavage. Control IgG did not show any effect on HK cleavage. (D) The effects of 3E8 antibody on DXS-induced bradykinin production were measured by ELISA. DXS induced a dramatic increase in bradykinin production, but 3E8 at 3.75 μM completely blocked DXS-induced bradykinin production. The 3E8 at lower concentrations (0.15 or 0.75 μM) also significantly blocked bradykinin production. Control IgG did not show any effects on DXS-induced bradykinin production. Data are denoted as mean ± SD. **P ≤ 0.01, and ***P ≤ 0.001. P > 0.05 was not significant (n.s.).

Fig. 2.
Fig. 2.

HK antibodies block Aβ42-induced HK cleavage and bradykinin release in human plasma. (A) Representative transmission electron microscopy image of Aβ42 used in this study. (Scale bar, 500 nM.) (B) HK Western blot shows that, in the absence of HK antibodies, Aβ42 completely cleaved HK. However, the 3E8 HK antibody dose-dependently blocked Aβ42-induced HK cleavage. The 2B7 antibody (at 0.1 and 0.5 μM) also showed a limited protective effect against HK cleavage. The 4B12 HK antibody and control IgG did not show any effects on HK cleavage. (C) The protective effects of 3E8 HK antibody were quantified as described in Fig. 1. The 3E8 antibody dose-dependently blocked Aβ42-induced HK cleavage, and, at 0.5 μM, it completely blocked HK cleavage. Control IgG did not show any effect on HK cleavage. (D) The effects of 3E8 antibody on Aβ42-induced bradykinin production were measured by bradykinin ELISA. Aβ42 induced a dramatic increase in bradykinin production, but the addition of 3E8 (0.5 μM) completely blocked Aβ42’s effects on bradykinin generation. The 3E8 at 0.02 or 0.1 μM also significantly blocked bradykinin production. Control IgG did not show any effects on Aβ42-induced bradykinin production. Data are denoted as mean ± SD. **P ≤ 0.01, ***P ≤ 0.001. P > 0.05 was not significant (n.s.).

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