β Adrenergic Receptor Kinase C-Terminal Peptide Gene-Therapy Improves β2-Adrenergic Receptor-Dependent Neoangiogenesis after Hindlimb Ischemia

After hindlimb ischemia (HI), increased catecholamine levels within the ischemic muscle can cause dysregulation of beta(2)-adrenergic receptor (beta(2)AR) signaling, leading to reduced revascularization. Indeed, in vivo beta(2)AR overexpression via gene therapy enhances angiogenesis in a rat model of HI. G protein-coupled receptor kinase 2 (GRK2) is a key regulator of beta AR signaling, and beta adrenergic receptor kinase C-terminal peptide (beta ARKct), a peptide inhibitor of GRK2, has been shown to prevent beta AR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury through restoration of beta(2)AR protective signaling (i.e., protein kinase B/endothelial nitric oxide synthase). Herein, we tested the potential therapeutic effects of adenoviral-mediated beta ARKct gene transfer in an experimental model of HI and its effects on beta AR signaling and on endothelial cell (EC) function in vitro. Accordingly, in this study, we surgically induced HI in rats by femoral artery resection (FAR). Fifteen days of ischemia resulted in significant beta AR down-regulation that was paralleled by an approximately 2-fold increase in GRK2 levels in the ischemic muscle. Importantly, in vivo gene transfer of the beta ARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and beta AR density in the ischemic muscle, compared with control groups. The effect of beta ARKct expression was also assessed in vitro in cultured ECs. Interestingly, ECs expressing the beta ARKct fenoterol, a beta(2)AR-agonist, induced enhanced beta(2)AR proangiogenic signaling and increased EC function. Our results suggest that beta ARKct gene therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced beta(2)AR down-regulation.