Impaired neoangiogenesis in β2-adrenoceptor gene-deficient mice: Restoration by intravascular human β2-adrenoceptor gene transfer and role of NFkB and CREB transcription factors

Background and Purpose: There is much evidence supporting the role of β ₂-adrenoceptors (β ₂AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of β ₂AR knock-out mice (β ₂AR-/-) in vivo and explored possible molecular mechanisms in vitro. Experimental Approach: Femoral artery resection (FAR) was performed in wild-type and β ₂AR-/-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 β ₂AR (ADβ ₂AR) was used to reinstate β ₂ARs in β ₂AR-/-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 β ₂AR-/-mice subjected to FAR, but was restored by gene therapy with ADβ ₂AR. The proangiogenic responses to a variety of stimuli were impaired in β ₂AR-/-EC in vitro. Moreover, removal of β ₂ARs impaired the activation of NFκB, a transcription factor that promotes angiogenesis; neither isoprenaline (stimulates βARs) nor TNFα induced NFκB activation in β ₂AR -/-EC. Interestingly, cAMP response element binding protein (CREB), a transcription factor that counter regulates NFκB, was constitutively increased in β ₂AR -/-ECs. ADβ ₂AR administration restored β ₂AR membrane density, reduced CREB activity and reinstated the NFκB response to isoprenaline and TNFα. Conclusions and Implications: Our results suggest that β ₂ARs control angiogenesis through the tight regulation of nuclear transcriptional activity.