The angiogenic response to Bradykinin "in vitro" : the role of Bradykinin receptors in hypoxic hearts and tumors

End organ damage resulting from hypertension is a leading
cause of morbidity and mortality worldwide. In
hypertension, left ventricular mass increases resulting in
left ventricular hypertrophy (LVH). LVH increases the risk
of heart failure and sudden cardiac death. This is due to
the decreased supply of oxygen and nutrients (ischemia) to
the myocardium because of vascular rarefaction. Research
has focused on inducers of angiogenesis such as basic
fibroblast growth factor and vascular endothelial growth
factor to improve myocardial oxygenation and function.
However, recently components of the Renin-Angiotensin-
Aldosterone System (RAAS), which contributes to blood
pressure control, have been shown to affect angiogenesis.
Angiotensin-converting-enzyme (ACE) inhibitors are used to
treat high blood pressure and congestive heart failure.
These block the conversion of physiologically inactive
angiotensin I to active vasoconstrictive angiotensin II and
inhibit the breakdown of Bradykinin (BK), a potent
vasodilator and mediator of inflammation. ACE inhibitors
increased capillary density in ischemic tissue by the
induction of new microvessels in ischemic rat limbs in
vivo. Several lines of evidence suggest Bradykinin to
possess significant angiogenic activity. Hence, Bradykinin
may mediate the effect of ACE inhibitors. Still, it is
unclear through whether Bradykinin promotes vascularization
of the ischemic heart via the Bradykinin receptor subtype 1
or 2. On the other hand, blocking angiogenesis could be a
strategy to arrest tumor growth, since tumor growth and
metastasis depend on angiogenesis. However, it is yet to be
fully elucidated whether and through which mechanisms
Bradykinin induces angiogenesis in tumors. Therefore, the aim of this thesis was in the first line to
clarify the angiogenic potential of Bradykinin in the
ischemic heart in vitro, especially the roles of the two
Bradykinin receptor subtypes in the regulation of
Bradykinin-induced angiogenesis. In second line, the thesis
aims to comparatively assess the role of Bradykinin and
requirement of Bradykinin receptors in cancer, i.e.
melanomas.
To do so, we used an in vitro model of angiogenesis of the
murine heart under moderate hypoxic conditions (3% O2).
Pilot experiments showed decreased angiogenic potential of
hypertrophied rodent hearts compared to normal healthy
controls. When using ACE inhibitors, angiogenesis in vitro
of hypoxic normal and hypertrophied hearts increased, and,
interestingly, Bradykinin showed a potent induction of
capillary like sprout formation.
This angiogenic effect was induced at low (10nM) but not at
high concentrations of Bradykinin (1mM). RT-PCR showed
expression of both Bradykinin receptor subtypes in hypoxic
mouse hearts. The angiogenic response to Bradykinin was
inhibited by a specific Bradykinin receptor 2 (BKR2)
inhibitor, but not by an inhibitor of Bradykinin receptor 1
(BKR1). A specific BKR1 agonist reduced angiogenesis.
Bradykinin-induced angiogenesis was not impaired in BKR1 (-
/-) mouse hearts. Different nitric oxide synthase
inhibitors (L-NAME, L-NIL, NIO) almost completely abrogated
the in vitro mouse heart angiogenesis response to
Bradykinin. Bradykinin did not induce angiogenesis in
hearts of iNOS (-/-) mice. Thus, in mouse hearts in vitro
Bradykinin at low nanomolar concentrations is angiogenic
under conditions of prolonged hypoxia. This angiogenic
effect is mediated by BKR2 activation and depends on iNOS. To assess the involvement of Bradykinin in cancer
angiogenesis, melanomas were injected and grown in the ear
of wildtype and BKR1 (-/-) mice, which acquired a BKR1 (-/-
) phenotype vasculature. In contrast to the findings in
hearts, we found that in melanomas from BKR1 (-/-) mice
angiogenesis in vitro was significantly lower as compared
to wildtype control. This suggests that melanomas in
contrast to hearts require vasculature with functional BKR1
to develop new microvessels.
In summary the key findings of this thesis are the
following: Bradykinin potently induces angiogenesis in
vitro of the hypoxic heart at nanomolar concentrations via
BKR2. At high Bradykinin concentrations or using specific
BKR1 agonists the angiogenic effect appears to be blocked.
Furthermore, functional iNOS is required for Bradykinin to
induce angiogenesis in vitro of the heart. In contrast to
the heart endothelial sprouting and angiogenesis, hypoxic
melanomas in vitro require BKR1.
Thus, specific stimulation of the BKR2 of the heart
vasculature may be a target to reduce tissue ischemia by
angiogenesis in the ischemic and/or hypertrophied heart.