Antagonism of Bradykinin B2 Receptor Prevents Inflammatory Responses in Human Endothelial Cells by Quenching the NF-kB Pathway Activation

<div><p>Background</p><p>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.</p><p>Methodology</p><p>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).</p><p>Principal findings</p><p>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.</p><p>Conclusion</p><p>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.</p></div

B2R blockade reduces BK-induced angiogenesis.

<p>(A) Representative pictures of pseudocapillaries formation in Matrigel from HUVEC in 0.1% FBS (a), exposed to BK (1 µM) (b), to fasitibant (1 µM) (c), to fasitibant+BK (d), observed 12 hrs after cell seeding. (B). Quantification of pseudocapillaries obtained by counting numbers of complete circles/well; Numbers represent mean ± SEM of three experiments run in triplicate. (C) BK induces vascularization in subcutaneously-injected Matrigel implants in mice. panel a: none, b: BK, c: fasitibant and d: fasitibant+BK. (D) Quantitative analysis of hemoglobin/angiogenesis in implants. For each condition (n = 6), the means ± SD are shown. **p<0.01, compared to untreated cells; ##P<0.01 to BK-treated cells.</p

Fasitibant suppresses BK-induced COX-2 signaling.

<p>(A–B) COX-2 and mPGES-1 expression (western blot) in HUVEC treated with BK (1 µM) for the indicated times. Gels are representative of three experiments. The ratio between COX-2 or mPGES-1 over actin is reported. *p<0.05, ***p<0.001, compared to untreated cells. (C) PGE-2 release in the conditioned medium of HUVEC treated with BK (1 µM) for the indicated times. All PGE-2 release experiments in this paper were performed in archidonic acid pre-treated cells. Numbers represent mean ± SEM of three experiments. *p<0.05, ***p<0.001, compared to untreated cells; (D) COX-2 and mPGES-1 expression in HUVEC treated with BK (1 µM, 6 hrs) with/without fasitibant (1 µM). Gels are representative of three experiments. Graphs represent the optical densities related to the ratio between COX-2 or mPGES-1 over actin. A.D.U. (arbitrary density unit), numbers represent mean ± SD of three experiments ***p<0.001, compared to untreated cells; ###P<0.001 to BK-treated cells. (E) PGE-2 release from HUVEC treated with BK (1 µM) in presence/absence of fasitibant (1 µM), for 8 hrs; Numbers represent mean ± SEM of three experiments. ***p<0.001, compared to untreated cells; ###P<0.001 to BK-treated cells; (F) COX-2 expression (western blot) in HUVEC pretreated for 30 min with IKK inhibitor VII (0.2 µM), and treated with BK (1 µM, 6 hrs). Gel is representative of three experiments. The ratio between COX-2 over actin is reported. ***p<0.001 compared to untreated cells; ###P<0.001 to BK-treated cells. (G) Representative pictures and quantification of pseudocapillary formation in Matrigel by HUVEC exposed to 0.1% FBS (panel a), BK (1 µM, panel b), IKK inhibitor VII (0.2 µM, panel c) with or without BK (1 µM, panel d), observed at 12 hrs after cell seeding. Quantification was obtained as above, ***p<0.001, compared to untreated cells; ###P<0.001 to BK-treated cells. Numbers represent mean ± SEM of three experiments.</p

BK-induced changes of endothelial junctions signals are blocked by fasitibant in HUVEC.

<p>(A) Confocal analysis of VEC expression (white arrowheads) in 0.1% FBS (a), BK (1 µM) (b), fasitibant (1 µM) (c), fasitibant+BK (d). (B–C) ZO-1 (60 X) and VEC (20 X) expression (white arrowheads), evaluated by immunofluorescence analysis, in 0.1% FBS (a), BK (1 µM) (b), fasitibant (1 µM) (c), fasitibant+BK (d). Bar = 20 µM. (D) Cytoplamic β-catenin phosphorylation, (western blot), in cells treated with BK (1 or 10 µM) with/without fasitibant (1 µM). Gels representative of three experiments; n = 3.</p

BK stimulates translocation/phosphorylation of NF-κB in circulating proangiogenic cells.

<p>(A) p65 (NF-κB) phosphorylation following exposure to BK (1 µM, 15 min) in presence/absence of fasitibant (0.1 µM). Gel are representative of three experiments. The ratio between p-p65 over p65 is reported. *p<0.05 compared to untreated cells; ###P<0.001 to BK-treated cells. (B) Immunofluorescence (40 X) of NF-κB translocation in PACs in 0.1% FBS (a), BK (1 µM) (b), fasitibant (0.1 µM,) (c), fasitibant+BK (d). Bar = 100 µM. (C) COX-2 expression in human hematopoietic progenitor cells pretreated for 30 min with IKK inhibitor VII (0.2 µM) or with fasitibant (0.1 µM), and treated with BK (1 µM, 6 hrs). Gel is representative of three experiments. The ratio between COX-2 over actin is reported. ***p<0.001 compared to untreated cells; ###P<0.001 to BK-treated cells. (D) PGE-2 release from PACs treated with BK (1 µM) in presence/absence of fasitibant (0.1 µM) or IKK inhibitor VII (0.2 µM), for 8 hrs. Numbers represent mean ± SEM of three experiments. *p<0.01, compared to untreated cells; #P<0.01 to BK-treated cells.</p