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-coniugated FITC-Dextran from upper to lower compartments (Numbers represent mean ± SEM of three experiments run in triplicate; n = 3); ***p<0.001, **p<0.01, *p<0.05 compared to untreated cells (D). Fasitibant (1 µM), prevents the enhanced permeability, n = 3; ***p<0.001 compared to untreated cells #p<0.05, ###p<0.001 to BK-treated cells.
Figure 2.
BK-induced changes of endothelial junctions signals are blocked by fasitibant in HUVEC.
(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.
Figure 3.
B2R blockade reduces BK-induced angiogenesis.
(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.
Figure 4.
Effect of fasitibant on angiogenesis and circulating proangiogenic cells in experimental osteoarthritis in rats.
(A) Representative images of hematoxylin/eosin staining (20 X) in synovial tissue from rats after intra articular injection of: saline (a), MIA (b), fasitibant with saline (c) or MIA with fasitibant, as described in Materials and Methods. Bar = 100 µM. Inset at higher magnification (40 X). Bar = 50 µM. (B–C) Representative images and quantification of CD-31 staining (40 X) in rats as described in A; Bar = 50 µM; quantification of CD-31 was performed counting 10 random field/section for slides; each slide has four sections. Data represent vessels counted for section in synovial tissue **P<0.01 compared to saline; ##P<0.01 compared to MIA. (D) ELISA immunoassay for VEGF in synovial fluids of rats treated as described in A. ***P<0.001 compared to saline; ##P<0.01 to MIA. (E) Representative images of CD-133 staining in rats as described in A; Bar = 50 µM. Inset at higher magnification (40 X).
Figure 5.
BK stimulates translocation/activation of NF-κB in HUVEC.
(A) NF-κB translocation following BK (1 µM) exposure for the indicated times, (B) or following exposure to BK (1 µM, 30 min) in presence/absence of fasitibant (1 µM). Gel are representative of three experiments. (C) Immunofluorescence analysis (40 X) of NF-κB translocation in HUVEC in 0.1% FBS (a), BK (1 µM) (b), fasitibant (1 µM,) (c), fasitibant+BK (d). Inset at higher magnification. Bar = 100 µM.
Figure 6.
Fasitibant suppresses BK-induced COX-2 signaling.
(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.
Figure 7.
BK stimulates translocation/phosphorylation of NF-κB in circulating proangiogenic cells.
(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.