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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 27, 2001 as doi:10.1096/fj.00-0503fje. |
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Department of Pharmacology, University of Florence, 50139, Florence, Italy;
* Institute of Pharmacological Sciences, University of Siena, Siena Italy; and
Microcirculation Research Institute and Department of Physiology, Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA
2Correspondence: Institute of Pharmacological Sciences, University of Siena, Via S. Piccolomini 170, 53100 Siena, Italy. E-mail: ziche{at}unisi.it
SPECIFIC AIMS
In this study we addressed the hypothesis that the contribution of bradykinin (BK) to angiogenesis depends on the type of receptor activated in endothelium and the ability to trigger an autocrine loop via the nitric oxide synthase (NOS) pathway. By the use of selective agonists and antagonists for the B1 and the B2 receptor, the effect of the kinin was assessed in vivo in the rabbit cornea, and the signaling cascades downstream receptor activation have been investigated on microvascular endothelial cells.
PRINCIPAL FINDINGS
1. The bradykinin/B1 receptor promotes angiogenesis in the rabbit
cornea
Gradient dismission of nanomolar concentration of BK (1
µg/pellet) into the cornea of albino rabbits induced a strong
angiogenic response in the absence of an inflammatory reaction. The B1
receptor agonist Lys-des-Arg9-BK reproduced BK
effect (Fig. 1a
, b
), whereas the B2 receptor agonist kallidin failed unless
higher doses of the peptide were used and inflammatory cells were
recruited into the corneal stroma. The effect of the B1 receptor
agonist proved to be specific since corelease of the selective BK/B1
receptor antagonist R715 into the corneal stroma completely blocked
B1-induced angiogenesis (0 positive implants of 4 performed), whereas
the selective BK/B2 receptor antagonist HOE140 exhibited little or no
effect.
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2. BK/B1 receptor up-regulates the NOS pathway in postcapillary
endothelium-promoting growth, whereas the BK/B2 receptor activates PLC
signaling but not cell proliferation
To assess the mechanisms underlying BK-induced angiogenesis, the
signaling cascade responsible for the angiogenic switch of endothelium
was studied in vitro. NOS activity and expression and phospholipase C
(PLC) activation were measured in cultured coronary postcapillary
venular endothelial cells (CVEC) exposed to BK and the selective B1 and
B2 receptor agonists. BK and the B1 receptor agonist increased by
2.6-fold endothelial NOS mRNA expression and activity (180±20% and
155±15% of basal NOS activity, respectively), whereas the B2 receptor
agonist did not. Treatment of CVEC with BK produced an increase of cGMP
accumulation (166±10% of basal cGMP levels). The selective inhibition
of B1 receptor blocked BK-induced cGMP accumulation and growth of
endothelial cells. The BK/B2 receptor, but not the B1 receptor,
promoted PLC-mediated IPs turnover and cytosolic
Ca2+ accumulation without affecting endothelial
cell growth (87±5% inhibition of BK-induced
Ca2+ accumulation in the presence of 1 µM B2
antagonist). These data indicated the existence of different
endothelial cell functions coupled to different BK receptors.
3. B1 receptor activation up-regulates FGF-2 in endothelium and
blockade of endogenous FGF-2 results in inhibition of BK/B1-induced
endothelium proliferation
During angiogenesis, NOS activity has been proved to up-regulate
fibroblast growth factor 2 (FGF-2). The role of endogenous FGF-2 in
transducing the angiogenic property of the BK/B1 receptor was then
assessed in CVEC. RT-PCR analysis of total RNA in CVEC exposed to 100
nM BK or to the selective B1 and B2 agonists evidenced a 2.6- and a
2-fold induction of FGF-2 mRNA expression in response to BK and to the
B1 agonist, respectively, whereas the B2 agonist was ineffective.
Accordingly, BK and Lys-des-Arg9-BK induced a
significant increase in the expression of 18 and 22–24 kDa FGF-2
isoforms that promoted cell proliferation (Fig. 2a
). In rabbit corneas where endogenous FGF-2 was
neutralized by the gradient dismission of anti-FGF-2 monoclonal
antibody, the angiogenic effect of BK/B1 receptor agonist was
completely prevented (Fig. 1a
, c
).
|
4. Activation of the NOS pathway by BK/B1 receptor is responsible
for endogenous FGF-2 up-regulation and growth of endothelial cells
To clarify the link among BK/B1 receptor, FGF-2, and
growth elevation in endothelium with NOS pathway activation, FGF-2
transcription and cell growth were assessed in CVEC where NO release
and function were neutralized by the NOS inhibitor L-NMMA or the
guanylate cyclase inhibitor ODQ. Inhibition of NO release resulted in
blockade of the BK/B1 receptor induced FGF-2 mRNA up-regulation (Fig. 2b
) and growth (Fig. 2c
), indicating that FGF-2
transcription was under the control of the NOS pathway. The mitotic
activity triggered by BK/B1 receptor consistently required both NO
synthesis and cGMP generation within the endothelial cell compartment
(Fig. 2c
), suggesting the existence of an autocrine loop
involving NO-cGMP production and FGF-2 up-regulation.
CONCLUSIONS
This work demonstrates that BK is a powerful angiogenic
stimulus in vivo and in vitro. The angiogenic effect of BK is due to
the direct activation of B1 receptor on capillary endothelial cells,
which transduces the autocrine up-regulation of FGF-2 by the endogenous
activation of the NOS pathway. BK/B2 receptor activation contributes to
angiogenesis via recruitment of inflammatory mediators, requires higher
tissue levels of BK, does not involve endothelial cell proliferation,
and is preferentially linked to PLC activation. Thus, it is concluded
that BK effects on angiogenesis are twofold, depending on the type of
receptor and the signal transduction pathway involved (Fig. 3
).
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Two subtypes of BK receptors, B1 and B2, have been defined by their
pharmacological properties. BK/B1 receptor functions are largely
unknown. Most of the BK effects reported are linked to B2 receptor
activation. Our data indicate that the B1 receptor is constitutively
expressed in coronary postcapillary venular endothelium and
its activation triggers the angiogenic switch of the endothelium via
the noninflammatory pathway. The proangiogenic events are independent
of B2 receptor activation and PLC signaling cascade. Thus, endothelial
cell functions other than proliferation must be ascribed to the B2
receptor, possibly linked to inflammatory cell recruitment and
modifications in permeability at the capillary level (Fig. 3)
.
We recently reported that BK potentiates FGF-2 effect in promoting endothelial cell growth. Our present findings indicate that the angiogenic program of BK/B1 receptor depends on the endogenous up-regulation of FGF-2, which exerts an autocrine control on endothelial cell growth. This demonstration highlights the relevance of the autocrine control exerted by capillary endothelial cells in the evolution of angiogenesis and the role of FGF-2 in supporting angiogenesis preferentially by paracrine/autocrine mechanisms. Since other peptides released during wound healing contribute to angiogenesis by up-regulating FGF-2 in endothelium and because of the preferential paracrine mode of action of FGF-2, the ability of endothelium to up-regulate FGF-2 can be regarded as a rate-limiting event in the cascade controlling angiogenesis.
Our data demonstrate that upstream signaling leading to FGF-2 up-regulation is controlled by the NOS pathway. Thus, as reported for other angiogenic peptides, the NOS pathway activation in endothelium sustains angiogenesis by priming endothelial cell in an autocrine/paracrine fashion. The existence of an autocrine loop within the capillary endothelium is thus reinforced by the fact that BK/B1 receptor induces NOS activation and cGMP accumulation, both required to transduce FGF-2 up-regulation and postcapillary endothelial cell proliferation. Nevertheless, characterization of molecular events at the nuclear level for the transcriptional control exerted by BK remains to be solved.
A sequence of complex interactions among positive and negative regulators contributes to the outcome of angiogenesis. Whether the process will result in the restoration of tissue function or rather in disrupted architectural organization of the tissue structure relies considerably on the type of molecules and cells involved into the process. The relative contribution of inflammatory mediators as well as their cellular targets can be of importance for the final outcome of angiogenesis toward restitutio ad integrum vs. fibrosis. The finding that BK contribute to angiogenesis depends on the type of receptor and the signaling activated at the capillary level is important for the development of therapeutical strategies. Although B2 receptors are attracting most interest at the clinical level, our data suggest that the targeting of B1 receptor might provide better results in angiogenesis-dependent diseases.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0503fje ; to cite this article, use FASEB J. (April 27, 2001) 10.1096/fj.00-0503fje 
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