Impaired neoangiogenesis in β₂-adrenoceptor gene-deficient mice: restoration by intravascular human β₂-adrenoceptor gene transfer and role of NFκB and CREB transcription factors.
OBJECTIVE
There is much evidence supporting the role of β₂-adrenoceptors (β₂AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of β₂AR knock-out mice (β₂AR-/-) in vivo and explored possible molecular mechanisms in vitro.


METHODS
Femoral artery resection (FAR) was performed in wild-type and β₂AR-/- mice and adaptive responses to chronic HL ischaemia were explored; blood flow was measured by ultrasound and perfusion of dyed beads, bone rarefaction, muscle fibrosis and skin thickness were evaluated by immunoflourescence and morphometric analysis. Intrafemoral delivery of an adenovirus encoding the human β₂AR (ADβ₂AR) was used to reinstate β₂ARs in β₂AR-/- mice. Molecular mechanisms were investigated in mouse-derived aortic endothelial cells (EC) in vitro, focusing on NFκB activation and transcriptional activity.


RESULTS
Angiogenesis was severely impaired in β₂AR-/- mice subjected to FAR, but was restored by gene therapy with ADβ₂AR. The proangiogenic responses to a variety of stimuli were impaired in β₂AR-/- EC in vitro. Moreover, removal of β₂ARs impaired the activation of NFκB, a transcription factor that promotes angiogenesis; neither isoprenaline (stimulates βARs) nor TNFα induced NFκB activation in β₂AR(-/-) EC. Interestingly, cAMP response element binding protein (CREB), a transcription factor that counter regulates NFκB, was constitutively increased in β₂AR(-/-) ECs. ADβ₂AR administration restored β₂AR membrane density, reduced CREB activity and reinstated the NFκB response to isoprenaline and TNFα.


CONCLUSIONS
Our results suggest that β₂ARs control angiogenesis through the tight regulation of nuclear transcriptional activity.
Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed Immunologiche, Università Federico II, Napoli, Italy.
Michele
M
Ciccarelli
Daniela
D
Sorriento
Ersilia
E
Cipolletta
Gaetano
G
Santulli
Anna
A
Fusco
Rui-Hai
RH
Zhou
Andrea D
AD
Eckhart
Karsten
K
Peppel
Walter J
WJ
Koch
Bruno
B
Trimarco
Guido
G
Iaccarino
D000818:Animals; D001783:Blood Flow Velocity; D050882:CREB-Binding Protein / Q000378:metabolism*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007511:Ischemia / Q000503:physiopathology* / Q000628:therapy; D008156:Luciferases / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism*; D018919:Neovascularization, Physiologic; D011869:Radioligand Assay; D018343:Receptors, Adrenergic, beta-2 / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D016328:NF-kappa B; D018343:Receptors, Adrenergic, beta-2; D042461:Vascular Endothelial Growth Factor A; D008156:Luciferases; D050882:CREB-Binding Protein