P595Inhibition of G-protein coupled receptor kinese 2 (grk2) by in vivo gene transfer enhances neoangiogenesis in a rat model of hindlimb ischemia

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Abstract

Purpose

Recent data have shown a relevant role of endothelial (EC) β2 adrenergic receptor (β2AR) in the modulation of in vivo angiogenesis. More precisely, following hindlimb ischemia (HI) this receptor is downregulated and its overexpression by in vivo gene transfer is able to enhance ischemia-induced neoangiogeneis. Since G-protein coupled receptor kinase 2 (GRK2) is a pivotal regulator of βAR signaling, inducing receptor desensitization/downregulation, we evaluated whether modulation of GRK2 activity might affect in vivo angiogenesis following HI.

Methods

At this aim, we surgically induced HI in rats and, at same time, we performed in vivo adenoviral-mediated gene transfer of GRK2 (to enhance GRK2 activity) or of the peptide βARKct (to inhibit GRK2 activity) to the endothelium of the rat femoral artery. 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 3,7,10,14 days post-surgery.

Results

Ultrasound showed impaired ischemic hindlimb perfusion in ischemic control groups (GFP and vehicle treated rats) compared with sham. GRK2 protein overexpression resulted in further reduction in ischemic hindlimb perfusion, whereas, βARKct expression restored blood flow in the ischemic limb compared with control 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 βARKct expression enhanced neo-capillarization in the ischemic hindlimb. Importantly, GRK2 overexpression enhanced β2AR downregulation at the plasma membrane of the ischemic muscle vs. controls, whereas βARKct restored β2AR density. In vitro cell monolayer-wounding assay, using bovine EC, showed enhanced EC migration after Adenovirus-βARKct infection, and reduced migration when GRK2 was overexpressed compared to GFP and not-infected ECs.

Conclusion

Our results suggest that modulation of GRK2 levels/activity has crucial effects on ischemia-induced angiogenesis, and indicate GRK2 inhibition as a novel therapeutic strategy in HI by preventing ischemia-induced β2AR downregulation.

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