J Cardiol Clin Res 4(2): 1057.
Submitted: 25 January 2016; Accepted: 02 March 2016; Published: 04 March 2016
Research Article
Bradykinin-Induced Shock Increase Exhaled Nitric Oxide, Complement Activation and Cytokine Production in Pigs
Knut Fredrik Seip1 Bjørg Evjenth2, Anders Hovland3, Knut Dybwik4, Harald Thidemann Johansen5, Hilde Fure6, Tom Eirik Mollnes6,7 and Erik Waage Nielsen8*
1Department of Pharmaceutical Chemistry, University of Oslo, Norway
2Department of Pediatrics, Nordland Hospital, Norway
3Division of Internal Medicine, University of Tromsø, Norway
4Department of Anesthesiology, University of Nordland, Norway
5Department of Pharmaceutical Biosciences, Norway
6Research Laboratory, Nordland Hospital, Norway
7Department of Clinical Medicine, University of Tromsø, Norway
8Department of Anesthesiology, University of Nordland, Norway
*Corresponding author: Erik Waage Nielsen, Department of Anesthesiology, University of Nordland, Nordland Hospital, Prinsens gate 8095 Bodø, Norway, Tel: 47-90788035;
Email: erikwn@me.com
Bradykinin is an important mediator in blood pressure regulation, ischemic
precondition and capillary leakage, allergy, anaphylaxis, inflammation, and
nociception, at least partly via the generation of nitric oxide (NO). Macrophages
are particularly abundant in the porcine lung circulation. Upon bradykinin binding
macrophages release cytokines and endothelial cells increase plasma leakage. Both
cells produce NO. The complement, hemostatic, fibrinolytic and kinin plasma cascade
systems crosstalk and interacts with many inflammatory systems. In the present study we
investigated the effect of the shock induced by intravenously infused bradykinin on the
cascade systems, cytokines, plasma leakage and exhaled NO in pigs. The metabolite of
bradykinin, BK1-5, was measured in plasma by a sensitive, specific and reliable liquid
chromatography-mass spectrometry method to verify exposure and in vivo metabolism
of bradykinin. We show for the first time in vivo how bradykinin exposure induced
shock and increased exhaled NO, activated complement and hemostasis and induced
cytokine production and capillary leakage. The results broaden our understanding
of how bradykinin activates endothelial cells and macrophages to induce shock and
inflammation. This should encourage further studies.
Keywords: Nitric oxide; Bradykinin; Macrophage; Endothelial cell; Plasma leakage; Complement activation; Coagulation; Cytokines; Porcine
Bradykinin is a potent nine amino acid short peptide
generated from kininogen in plasma. Bradykinin is involved
in blood pressure regulation, capillary leakage and ischemic
preconditioning of the heart [1]. Bradykinin also mediates
allergy, anaphylaxis, inflammation and nociception [2].
Bradykinin itself has an extremely short half-life (<30 s) due to
breakdown by angiotensin converting enzyme (ACE) and other
exopeptidases present in plasma. It is therefore difficult to detect
in vivo [3]. The kinin system, the intrinsic pathway of coagulation,
the complement system and the fibrinolytic system all cross-talk
[4]. Macrophages and endothelial cells release nitric oxide (NO)
and bradykinin markedly increase NO from endothelial cells
[5]. Ovine and porcine lung parenchyma have large amounts
of macrophages in their circulation, and the macrophage
densities are similar to that of Kupfer cells in the rat liver [6].
More than 25 years ago, bradykinin and its metabolite des Argbradykinin
were shown to stimulate murine macrophages to
release tumor necrosis factor (TNF) and interleukin-1 (IL-1) [7].
The quantitative relation between bradykinin and its effects on
circulation and inflammation are largely unknown in all species.
Therefore, we infused pigs with bradykinin to obtain shock and
investigated the effects of bradykinin on exhaled NO, the plasma
cascades and inflammation.
Study design
In this porcine study we administered bradykinin in three
different ways. Animal A was given a continuing and steadily
increasing infusion of bradykinin with bolus doses late in
the experiment. Animal B was exposed to a high bolus dose of
bradykinin early in the experiment. Animal C was pretreated with
1.5 mg captopril (ACE-inhibitor) prior to infusion of bradykinin.
In all the pigs, the infusion of bradykinin was adjusted to maintain
a critically low mean arterial pressure (MAP) of 40 mmHg.
Animals
Three Norwegian Landrace pigs of either sex (23 kg ±2) were
treated in accordance with the Norwegian laboratory animal
regulations and the study was approved by the Norwegian Animal
Research Authority (FOTS #2843). Housekeeping, anesthesia,
surgery and euthanasia were performed as previously described
[8]. Ringer´s acetate was given intravenously at 10 ml/kg/h.
Monitoring and examinations
Electrocardiography, end-tidal CO2, pulse oximetry, regular
interval blood gas, systemic intra-arterial blood pressure
and pulmonary artery pressure were recorded as described
previously [8]. Basic echocardiographic parameters were
obtained by a Vivid S6 scanner (GE Vingmed, Horten, Norway)
with a 1.5-3.6 MHz matrix array probe. All echocardiographic
values are an average of five consecutive measurements with
standard deviations.
Nitric oxide in exhaled gas
NO was measured online by the multiple breath method with
a chemiluminescence analyzer, as described elsewhere [9,10].
Laboratory analyses
Cytokines, hemostatic markers and the terminal complement
complex (TCC) were measured in blood samples at baseline and
during bradykinin infusion by methods described previously
[11].
Measurements of bradykinin and BK1-5
Measurement of the stable bradykinin metabolite BK1-5 was
performed by a previously described liquid chromatographymass
spectrometry (LC-MS/MS) method [12]. Quantification of
bradykinin was performed by a commercial ELISA-kit from Uscn
Life Science Inc (Wuhan, China) that was validated by measuring
the internal standard of bradykinin.
Bradykinin increased exhaled NO and induced shock and capillary leakage
Bradykinin dose dependently increased exhaled NO (Figure
1). Even though a large decrease in MAP was seen, mean
pulmonary artery pressure (MPAP) remained constant (20-30
mmHg) throughout the infusion. Systemic vascular resistance
index (SVRI) was reduced by 46% in animal A, 65% in animal B
and 23% in animal C. A 37% and 30% fall in albumin was seen
in animal B and C, respectively, but not in animal A. A complete
echocardiographic assessment of left ventricular dimensions in
animal A showed 2.4 cm (+/- 0.2 cm) end systolic parasternal
long axis at baseline and 0.3 cm (+/- 0.1 cm) at peak bradykinin
infusion.
Bradykinin activated both complement and hemostasis and induced cytokine production
Inflammatory and hemostatic markers, including TCC,
thrombin-antithrombin complex (TAT), plasminogen activator
inhibitor-1 (PAI-1), IL-1β, TNF, interleukin-6 (IL-6) and
interleukin-8 (IL-8), were measured during the experiments.
After bradykinin infusion, TCC, TNF and IL-6 increased markedly
in Animal A, while all markers rose in Animal B (Figure 2). Animal
C developed an uncontrollable hypotension and died too early to
deliver data on these markers.
Massively increased BK1-5 measured by LC-MS/MS
After 60 minutes, animal A and B showed substantially
elevated levels of BK1-5. The signals were about two orders of
magnitude above the detection method’s validated maximum
(17.7 nmol/mL). Animal C, which was given an ACE-inhibitor
before bradykinin, had lower (15.8 nmol/mL), but elevated levels
of BK1-5 after 30 minutes of bradykinin infusion. The baseline
levels were below the method’s detection limit (35.4 pmol/mL)
for all the animals. ELISA measurements of bradykinin were
done in animal A, but failed to show the extensive exposure to
bradykinin as they never reached the lowest standard of 24 pg/
ml, which is consistent with its short half-life.
In this study, effects of intravenously infused bradykinin in
the absence or presence of an ACE-inhibitor was investigated
in pigs. We found an increase in exhaled NO, activation of
complement and hemostasis, increased cytokine production and
capillary leakage.
In all the animals, there was a clear link between the infused
amount of bradykinin and a severe lowering of MAP, whereas
MPAP remained unaffected. The sizeable reduction in end systolic
diameter of the left ventricle during peak infusion also indicates
that bradykinin reduces afterload and increases contractility.
This is consistent with earlier findings on the hemodynamic
effects of bradykinin infusion [13], and bradykinin’s known
activity as a vasodilator [14]. Our results also fit well with an
early study of bradykinin infusion to humans [15], and suggest
a different effect of bradykinin in the pulmonary circulation than
in the systemic circulation. It is tempting to speculate that the
high concentration of ACE on pulmonary endothelium protects
the lungs from bradykinin induced edema. This could also
explain why pulmonary edema is rare in patients with hereditary
angioedema who execessively produce bradykinin [16].
By measuring the NO concentration in expiration gas, the
effects of bradykinin could be indirectly monitored. A close
and rapid relation between exhaled NO and rate of bradykinin
infusion was seen (Figure 1). Whether bradykinin released NO
directly or indirectly via preformed cytokine cell stores, remains
to be investigated. However, the slow and moderate increase of
cytokines we measured suggest bradykinin as dominant directly.
Elevated exhaled NO after infused bradykinin is, to our knowledge,
for the first time observed and documented in the present study.
The increased exhaled NO was related to a quantifiable shock.
Stimulation of the B2-receptor by bradykinin is known to release
plasminogen-activator and produce NO and prostaglandin I2(PGI2
) from the endothelial cells [14,17,18]. Upon stimulation by
lipopolysaccharides macrophages per cell release ten times more
NO than do endothelial cells upon exogenously added bradykinin
[5]. The enzyme endothelial NO synthase (eNOS) is released
within seconds upon receptor stimulation and could explain the
rapid NO response [19]. A difference in exhaled NO was seen
between animal A and B (Figure 1). This should be expected as
animal A and B received bradykinin infusions at varying rates.
The difference may suggest a desensitizing effect by prolonged
and gradually increased exposure to bradykinin. A previous
publication also found desensitization after internalization of the
bradykinin-B2 receptor complex [20].
Figure 1 Effects of bradykinin infusion on exhaled NO.
Animal A: The infusion of bradykinin was gradually increased with a corresponding increase in exhaled NO. MAP fell below 40 mmHg after 30 minutes and was maintained at a low level as the infusion rate was kept constant.
Animal B: An initial bolus dose of bradykinin was given (initial peak) which induced a substantial increase in exhaled NO. The infusion rate was quickly escalated after the initial bolus and the levels of exhaled NO closely followed the infusion rate and remained at a relatively high level throughout the experiment. In this case, MAP dropped below 40
mmHg after 15 minutes.
Animal C: Captopril was given before bradykinin and exhaled NO quickly increased to more than twice the initial concentration after bradykinin infusion was started. The animal died after only 34 minutes after a large fall in MAP. BK inf. Bradykinin infusion, FENO: fractional exhaled nitric oxide, ppb: parts per billion.
Figure 1 Effects of bradykinin infusion on exhaled NO.
Animal A: The infusion of bradykinin was gradually increased with a corresponding increase in exhaled NO. MAP fell below 40 mmHg after 30 minutes and was maintained at a low level as the infusion rate was kept constant.
Animal B: An initial bolus dose of bradykinin was given (initial peak) which induced a substantial increase in exhaled NO. The infusion rate was quickly escalated after the initial bolus and the levels of exhaled NO closely followed the infusion rate and remained at a relatively high level throughout the experiment. In this case, MAP dropped below 40
mmHg after 15 minutes.
Animal C: Captopril was given before bradykinin and exhaled NO quickly increased to more than twice the initial concentration after bradykinin infusion was started. The animal died after only 34 minutes after a large fall in MAP. BK inf. Bradykinin infusion, FENO: fractional exhaled nitric oxide, ppb: parts per billion.
×
Both NO and PGI2 relax smooth muscles in the arterial wall
and systemic vascular resistance drops accordingly. The shock
we observed mimicked severe sepsis, and bradykinin mediates
inflammation [8]. A fall in albumin, as we observe during
bradykinin infusion, is also seen during sepsis [8]. It supports
the role of bradykinin as a mediator of capillary leakage in these
conditions. In a recent study exogenously infused bradykinin to
rats reduced the tight junction proteins claudin-5 and occludin
and increased plasma leakage. The mechanism in this brain
tumor model was up regulation of the eNOS and neuronal NOS
and the transcriptional repressor ZO-1 associated nucleic acid
binding protein ZONAB [21]. In macrophages the inducible NOS
(iNOS) is the key inflammatory enzyme and produce such high
levels of NO that in one study setting upon LPS administration
to mice genetically defect of iNOS, the fall in blood pressure
was markedly attenuated and early death averted [22].
Consequently, the relative contribution by macrophages and
endothelial cells to the capillary leakage and NO-production
we observed after bradykinin infusion is difficult to ascertain.
The presence of approximately 4000 bradykinin receptors per
guinea pig macrophage [23], and the reciprocal activation by NO
and cytokines on macrophages and endothelial cells gives the
impression that both cell types contribute to the results in our
study.
The inflammatory markers TCC, TNF and IL-6 were markedly
increased after bradykinin infusion (Figure 2). Interestingly, in
animal B (Figure 2), where a large bolus dose of bradykinin was
instantly followed by a large increase in exhaled NO and a marked
drop in MAP, these increases were several orders of magnitude
higher than in animal A. A marked increase in coagulation,
represented by TAT and PAI-1 was also present. It is possible that
this massive generation was a result of the very early bradykinin
bolus. It also suggests that bradykinin might be better tolerated
when its infusion rate is gradually increased. This also fits
well with the previously mentioned desensitizing theory from
prolonged bradykinin exposure. Even more interesting is the
possibility of a direct cross-talk between the kinin system on one
side and the complement and coagulation pathways on the other.
To our knowledge, we present here the first in vivo evidence that
bradykinin alone activates these cascade systems directly
Figure 2 Effects of bradykinin on inflammatory markers and complement activation.
Several markers of inflammation and TCC for two of the three animals (A and B) are shown. Animal A: A moderate increase in TCC, TNF and IL-6 was observed after 60 minutes of bradykinin infusion and a further increase was measured after 115 minutes of bradykinin infusion.
Animal B: A substantial increase (note the logarithmic scale) in all the measured cytokines was observed after 60 minutes of bradykinin infusion. Except for PAI-1, no further increase was seen after 90 minutes of bradykinin infusion. TAT: thrombin-antithrombin complex, PAI-1: plasminogen activator inhibitor-1, IL-1β: interleukin-1β, TNF: tumor necrosis factor, IL-6: interleukin-6 and IL-8: interleukin-8.
Figure 2 Effects of bradykinin on inflammatory markers and complement activation.
Several markers of inflammation and TCC for two of the three animals (A and B) are shown. Animal A: A moderate increase in TCC, TNF and IL-6 was observed after 60 minutes of bradykinin infusion and a further increase was measured after 115 minutes of bradykinin infusion.
Animal B: A substantial increase (note the logarithmic scale) in all the measured cytokines was observed after 60 minutes of bradykinin infusion. Except for PAI-1, no further increase was seen after 90 minutes of bradykinin infusion. TAT: thrombin-antithrombin complex, PAI-1: plasminogen activator inhibitor-1, IL-1β: interleukin-1β, TNF: tumor necrosis factor, IL-6: interleukin-6 and IL-8: interleukin-8.
×
The BK1-5 measurements showed a massive signal after
infusion of bradykinin (possibly more than 100 000 times
increase compared to baseline levels), whereas measurements of
bradykinin by ELISA did not reveal any changes in concentration.
This illustrates the very rapid metabolism of bradykinin [3].
The significance was further exemplified in animal C. Here the
increase in BK1-5 was modest, as the conversion of bradykinin
into BK1-5 is hampered by inhibition of ACE [24]. Bönner et al.
found that an ACE-inhibitor potentiated a bradykinin infusion
20-fold [15]. This is in line with our observations in animal
C. Although the bradykinin infusion in animal C was reduced
compared to animal A and B, the presence of an ACE-inhibitor
caused a massive bradykinin buildup and a rapid circulatory
collapse. It also explains why the exhaled NO in animal C reached
the highest value in our experiment.
This in vivo study shows for the first time how infused
bradykinin induced shock and markedly increased exhaled
NO, activated the coagulation and complements system and
induced several pro-inflammatory cytokines. The rise in these
markers was related to the stable bradykinin metabolite BK1-
5 with a sensitive LC-MS/MS method. These results suggest
that bradykinin directly activates macrophages and endothelial
cells and contributes to cytokine response, plasma leakage and
increased exhaled NO. Our study expands the understanding of
bradykinin’s many roles in vivo.
This study was financially supported by The Norwegian Council on Cardiovascular Disease and the European Community’s Seventh Framework Programme under grant
agreement n°602699 (DIREKT).
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Cite this article: Seip KF, Evjenth B, Hovland A, Dybwik K, Johansen HT, et al. (2016) Bradykinin-Induced Shock Increase Exhaled Nitric Oxide, Complement Activation and Cytokine Production in Pigs. J Cardiol Clin Res 4(2): 1057.
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