In Vitro Modeling of Bradykinin-Mediated Angioedema States

Abstract

Kinins (peptides related to bradykinin, BK) are formed from circulating substrates, the kininogens, by the action of two proteases, the kallikreins. The only clinical application of a BK receptor ligand, the B₂ receptor antagonist icatibant, is the treatment of the rare hereditary angioedema (HAE) caused by the deficiency of C1-esterase inhibitor (C1-INH). Less common forms of HAE (genetic variants of factor XII, plasminogen, kininogen) are presumably mediated by increased BK formation. Acquired forms of BK-mediated angioedema, such as that associated with angiotensin-I converting enzyme (ACE) inhibition, are also known. Antibody-based analytical techniques are briefly reviewed, and support that kinins are extremely short-lived, prominently cleared by ACE. Despite evidence of continuous activation of the kallikrein-kinin system in HAE, patients are not symptomatic most of the time and their blood or plasma obtained during remission does not generate excessive immunoreactive BK (iBK), suggesting effective homeostatic mechanisms. HAE-C1-INH and HAE-FXII plasmas are both hyperresponsive to fibrinolysis activation. On another hand, we suggested a role for the alternate tissue kallikrein-kinin system in patients with a plasminogen mutation. The role of the BK B₁ receptor is still uncertain in angioedema states. iBK profiles under in vitro stimulation provide fresh insight into the physiopathology of angioedema.

Keywords: acquired angioedema; analytical techniques for kinins; bradykinin; hereditary angioedema; kallikrein–kinin system.

Figure 1

Schematic representation of the kallikrein-kinin system, featuring the 2 validated pathways of kinin generation, that of plasma kallikrein (part of the contact system) releasing bradykinin (BK) from high molecular weight kininogen (HK) and that mediated by secreted tissue kallikrein (KLK-1), generating Lys-BK mainly from low molecular weight kininogen (LK). Some of the known mutant genes that cause HAE-nC1-INH (dark red background with dotted arrows pointing to the corresponding mutant proteins) and the standardized stimuli applied in vitro to trigger kinin formation (pale orange background) are also indicated. Elements of the contact system are represented in light blue. Black and blue arrows represent biochemical reactions. Green arrows indicate an agonist effect on a receptor. “⊥” Inhibition of a protease or peptidase. See Table 1 for abbreviations.

Figure 2

Cross-reactivity of the BK EIA with selected kinin sequences. The inactive metabolite des-Arg¹-BK (= BK fragment 2–9) exhibits full cross-reactivity vs. BK. N-terminal extended sequences, like Arg-BK (shown here), or Lys-BK are also fully reactive. Truncated sequences at the C-terminus, like the optimal B₁ receptor agonist Lys-des-Arg⁹-BK, do not cross react at all [12]. An extended sequence like Arg-BK-Arg-Arg, an inactive prodrug releasing active kinins via the action of carboxypeptidases [31], has a partial reactivity, showing the importance of a free COOH terminus. Reproduced in part from [12].

Figure 3

(A) Degradation of synthetic BK (100 nM) added to human plasma from healthy subjects in the presence or absence of the ACE inhibitor enalaprilat. Samples were incubated at 37 °C under agitation for the indicated time periods before extraction and EIA determination of BK. The number of replicated from different healthy donors is indicated by “n”. In the presence of the ACE inhibitor, the data are compatible with a first order decay with a half-life of 7.1 min, whereas BK was completely cleared in 5 min the absence of enalaprilat (t_1/2 34 s). Reproduced from [18]. (B) Catabolism of BK, measured as [iBK], by intact HUVECs as a function of time. Values are means ± s.e.m. of 3 determinations. A first order decay model has been applied (t_1/2 values reported in main text). BK added to similar wells without cells (nude plastic) was stable when incubated in an identical manner (data not shown).

Figure 4

Effect of incubation under agitation in Hank’s balanced salt solution containing BK (500 nM) on adherent HUVECs (matched phase contrast and red fluorescence fields, original magnification 100×). Propidium iodide (PI) staining was performed as a test of viability (positive control, Triton X100, 0.5%). Total counted cell numbers are given next to the histograms.

Figure 5

Kinetics of iBK concentrations as a function of time and stimulation in samples of normal plasma incubated at 37 °C in the presence of enalaprilat (130 nM). Panels represent different forms of in vitro stimulation added to plasma at time zero: (A) control; (B) KLK-1 10 nM; (C) Kontact-APTT™ 2% v/v; (D) tPA 169 nM. The data is from the fusion of the 2 control groups (healthy volunteers) from [18,44]. The average age is 45.9 and 12 females are represented in this group of 16 subjects. Indications of significant variations induced by specific HAE subtypes are indicated by red arrows. Measurements made from a partly clotted plasma sample are also shown: in the presence of enalaprilat but no other stimulant, the sizeable concentration of BK at time zero decreases according to a first-order elimination kinetics (panel A, t_1/2 12.3 min, inset).