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

Abstract

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.