Abstract 15873: Modulation of Ischemic Neoangiogenesis by in vivo Gene Transfer and Manipulation of G Protein-Coupled Receptor Kinase-2 Activity: A Novel Role for the Peptide BARKct
Recently, a crucial role of beta2 adrenergic receptor (B2AR) in endothelial cell (EC) function has been reported, with relevant role in in vivo angiogenesis. Importantly, following hindlimb ischemia (HI) this receptor is downregulated. Since G-protein coupled receptor kinase 2 (GRK2) is a crucial regulator of BAR signaling, inducing receptor desensitization/downregulation, we evaluated whether modulation of GRK2 activity might affect in vivo angiogenesis following HI. To this end, we surgically induced HI in rats. We used in vivo adenoviral-mediated gene transfer of GRK2 (to enhance GRK2 activity) or of the peptide BARKct (to inhibit GRK2 activity) to the endothelium of the rat femoral artery, at the time of surgery. Adenovirus (Ad) encoding for green fluorescent protein (GFP) or vehicle served as controls. Sham animals were included in the study. Transgene expression was confirmed 15 days post-surgery by western blotting and immunohistochemistry. Blood flow was measured by echo-doppler before, immediately after, and 3,7,14 days post-surgery. Ultrasound showed impaired ischemic hindlimb perfusion in ischemic control groups compared with sham. GRK2 protein overexpression resulted in further reduction in ischemic hindlimb perfusion, whereas, BARKct expression restored blood flow in the ischemic limb compared with controls rats. Dyed beads perfusion assay confirmed the echo results. Capillary density was reduced in the ischemic hindlimb of controls, and a further significant decrease was observed in GRK2 treated group, whereas BARKct expression enhanced neoangiogenesis in the ischemic hindlimb. Importantly, GRK2 overexpression enhanced B2AR downregulation at the plasma membrane of the ischemic muscle vs. controls, whereas BARKct restored B2AR density. In vitro cell monolayer-wounding assay, using bovine EC, showed enhanced EC migration with Ad-BARKct infection, and reduced migration when GRK2 was overexpressed compared to GFP and not-infected EC. Taken together our data suggest that modulation of GRK2 levels/activity has crucial effects on ischemia-induced angiogenesis, and suggest GRK2 inhibition as a novel therapeutic strategy in HI by preventing ischemia-induced B2AR downregulation
- © 2011 by American Heart Association, Inc.