Bradykinin (BK) induces angiogenesis by promoting vessel permeability, growth and remodeling. This study aimed to demonstrate that the B2R antagonist, fasitibant, inhibits the BK pro-angiogenic effects.
We assesed the ability of fasibitant to antagonize the BK stimulation of cultured human cells (HUVEC) and circulating pro-angiogenic cells (PACs), in producing cell permeability (paracellular flux), migration and pseocapillary formation. The latter parameter was studied in vitro (matrigel assay) and in vivo in mice (matrigel plug) and in rat model of experimental osteoarthritis (OA). We also evaluated NF-κB activation in cultured cells by measuring its nuclear translocation and its downstream effectors such as the proangiogenic ciclooxygenase-2 (COX-2), prostaglandin E-2 and vascular endothelial growth factor (VEGF).
HUVEC, exposed to BK (1–10 µM), showed increased permeability, disassembly of adherens and tight-junction, increased cell migration, and pseudocapillaries formation. We observed a significant increase of vessel density in the matrigel assay in mice and in rats OA model. Importantly, B2R stimulation elicited, both in HUVEC and PACs, NF-κB activation, leading to COX-2 overexpression, enhanced prostaglandin E-2 production. and VEGF output. The BK/NF-κB axis, and the ensuing amplification of inflammatory/angiogenic responses were fully prevented by fasitibant as well as by IKK VII, an NF-κB. Inhibitor.
This work illustrates the role of the endothelium in the inflammation provoked by the BK/NF-κB axis. It also demonstates that B2R blockade by the antaogonist fasibitant, abolishes both the initial stimulus and its amplification, strongly attenuating the propagation of inflammation.
The inflammation elicited by bradykinin (BK) through the B1 and B2 receptors (B1R, B2R) recapitules the cardinal signs of an inflammatory response as it induces: vascular permeability, hyperthermia, oedema, pain and neo-vessel formation (angiogenesis) [1]–[7]. More recently, BK has been described to be involved in the pathogenesis of degenerative joint diseases, such as the knee osteoarthritis [8]–[10]. During the osteoarthritis process, chronic inflammation promotes the imbalance of metabolic and degradative signals. BK, through the B2R, contributes to the chronic inflammatory response in the knee osteoarhritis, activating different cells, including synovial cells or chondrocytes, and inducing the release of pro-inflammatory cytokines, as well as the products of ciclooxygenase (COX) and lipooxygenase (LOX) [10], [11]. Several peptide and non-peptide B2R antagonists have been synthesised [12], [13]. Icatibant, a peptide compound, is one of the first B2R antagonists synthesised, now approved for the therapy of hereditary angio-oedema attacks [7], [14]. Recently, the non-peptide B2R antagonist fasitibant (formerly MEN16132) showed a remarkably high affinity and antagonist potency toward B2R in different species, including humans [15]–[18]. In preclinical models of inflammation and pain, including osteoarthritis, fasitibant was effective and long lasting in blocking both exogenous and endogenous BK [19]–[22]. The compound is now undergoing a phase II clinical study in knee osteoarthritis patients (ClinicalTrials.gov: NCT01091116).
Angiogenesis plays a pivotal role in the advancement of inflammatory diseases progression, including osteoarthritis, as a source of inflammatory cells, cytokine and protease activity [23].
Vascular growth both in the synovium and at the osteochondral junction have been associated with osteoarthritis, which is characterized by synovitis and progressive cartilage degeneration, thus novel therapies capable to limit angiogenesis, besides inflammation and pain, might be a desirable target [24], [25]. Notably, in the progression of osteoarthritis, the benefits of agents that suppress neovascularization has been very impressive, providing a solid rationale for pursuing anti-angiogenesis strategies in patients affected by chronic inflammatory diseases [26]. Of relevance to this study are recent reports describing the role of the kallikrein/bradykinin system, through the B2R in the recruitment of circulating pro-angiogenic cells, a process which leads to tissue vascularization [27].
BK is known to induce angiogenesis by activating endothelial cells, and promoting vessel permeability, growth and remodeling [28], [1], [4], [5]. The present study aimed to demonstrate that the B2R antagonist, fasitibant, inhibits the BK pro-angiogenic effects, both in in vitro and in vivo studies. Moreover, we provide evidence that, in endothelial cells and in circulating proangiogenic cells (PACs), BK activates the pro-inflammatory NF-κB transcriptional factor, which, in turn, promotes the overexpression of a wide array of inflammatory genes (e.g. interleukins, chemokines, COX-2, MMPs). As a measure of the functional NF-κB activation, we assessed the COX-2/prostaglandin E-2 (PGE-2) pathway, because of their established role as pro-inflammatory and pro-angiogenic signals. We demonstrate that fasitibant abolished pro-angiogenic effects by suppressing the B2R-dependent BK-induced NF-κB transcription factor activation.
In order to evaluate the anti-angiogenic activity exerted by fasitibant on cultured human umbelical venular endothelial cells, HUVEC, first, we assessed the presence of the BK receptors, B1 and B2, by measuring their respective mRNA and protein expression (Fig. 1A and B). The B2R was revealed in terms of messenger (Fig. 1A) and protein, and it was not modulated by the presence of BK (Fig. 1B and Fig. S1). The B1R was undetectable in HUVEC (Fig. 1A), either in basal condition (0.1% FCS) after BK or 10% FCS stimulation (Fig. S1).
Figure 1. B2R stimulation promotes changes of cell permeability.
(A) mRNA expression for B1 and B2 receptors, (B2R mRNA is approximately 339 pb) and (B) western blot analysis of B2 receptor in HUVEC. (Experiments are run three time; n = 3). (C) Permeability in HUVEC monolayer was detected as passage of fluorescence-coniu
